Postsynaptic proteins

ABSTRACT

A protein A that binds to the N-methyl-D-aspartate (NMDA) receptor and a protein B that interacts with protein A are provided. Furthermore, based on the marked promotion of the NMDA receptor mediated signal transduction by these proteins, providing a controlling agent (an inhibiting agent or a promoting agent) and a controlling method for the expression and/or the function of these proteins makes possible the elucidation of diseases caused by an anomaly in the NMDA receptor mediated signal transduction or in memory recall, including such neurodegenerative diseases as Alzheimer&#39;s disease, Parkinson&#39;s disease and polyglutamine disease, and allows for the prevention, improvement or treatment thereof.

TECHNICAL FIELD

The present invention relates to a postsynaptic density protein(hereinafter referred to as protein A), which possesses a PDZ domain andis capable of forming a complex with an N-methyl-D-aspartate(hereinafter abbreviated as NMDA) receptor; and a protein (hereinafterreferred to as protein B), which interacts with protein A; as well as acontrolling agent (an inhibiting agent or a promoting agent) of an NMDAreceptor mediated signal transduction, which inhibits or promotes theformation of the complex of protein A and the NMDA receptor; acontrolling agent of NMDA receptor mediated signal transduction, whichinhibits or promotes the interaction of protein A with protein B; amethod for controlling an NMDA receptor mediated signal transduction byinhibiting or promoting the formation of the complex of protein A andthe NMDA receptor; and a method for controlling an NMDA receptormediated signal transduction by inhibiting or promoting the interactionof protein A with protein B. More specifically, it relates to apolypeptide or peptide having complete or a partial amino acid sequenceof protein A or protein B; a polynucleotide containing a nucleotidesequence coding for the polypeptide or peptide, or containing thecomplementary nucleotide sequences thereof; a recombinant vectorcontaining the polynucleotide; a transformant containing the recombinantvector; an antibody directed against the polypeptide or peptide; acompound that interacts with the polypeptide or peptide; apharmaceutical composition containing one or more of the above; a methodfor manufacturing the polypeptide or peptide; a method for identifyingcompounds that interact with the polypeptide or peptide; a method formeasuring the polypeptide or peptide; and a reagent kit.

TECHNICAL BACKGROUND

A postsynaptic density protein (hereinafter abbreviated as PSD) formsthe postsynaptic densities that are present in postsynaptic cells andreceive the synaptic information during signal transduction betweennerve cells. In the postsynaptic membrane, PSD is involved in thelocalization and accumulation of functional membrane proteins (such asreceptors and ion channels), or the formation of bio-multisomes.

Meanwhile, proteins (that are involved in biological functions such asthe formation and maintenance of complex cell membrane structures (suchas synapses and tight junctions), the accumulation of membrane protein,signal transduction, the formation of complexes and the maintenance ofcell polarity) were reported to possess a peptide-binding domain thoughtto be involved in these functions as a module that bears protein-proteininteractions (Non-Patent Reference 1). This domain is called a PDZdomain and comprises 90 to 100 amino acid residues. Among thesebiological functions, the PDZ domain is thought to play a more importantrole in functions such as localizing a specific protein to a specificintracellular site, or promoting the binding of a protein containing thePDZ domain to a target protein. For example, involvement of the PDZdomain in the localization of signal transduction proteins to proteincomplexes in the cell membrane may be necessary to achieve appropriatesignal transduction.

In protein-protein interactions, the PDZ domain recognizes theC-terminal amino acid sequence of a target protein. The target proteinis often a transmembrane receptor or channel. For example, PSD-95 andPSD-93 (chapsyn 110 or KAP-5), which belong to the MAGUK protein(membrane associated guanylate kinase protein) family that is present inthe synapse of vertebrates or invertebrates, and hdlg (a human homologof Drosophila dig protein) have been reported to recognize theC-terminus of an NMDA receptor and the Shaker-type potassium ion channelKir 1.4, through their second and third PDZ domains (Non-PatentReference 2).

By analyzing the in vivo localization of proteins that possess the PDZdomain, and the localization of target proteins of the transmembranetype that have been identified by in vitro biochemical analyses, it wasdemonstrated that PSD-95, PSD-93, and hdlg all showed the samelocalization as an NMDA receptor, or a potassium ion channel, in eachtissue as well as in each cell species of the nervous system (Non-PatentReference 3, Non-Patent Reference 4, Non-Patent Reference 5, andNon-Patent Reference 6).

In addition, when PSD-95 is co-expressed in fibroblasts with the NMDAreceptor or a potassium ion channel, clustering of these proteins on thecell surface is observed, while when they are each expressed alone, onlya diffuse localization is observed (Non-Patent Reference 4). Meanwhile,the splicing isoform, which lacks the C-terminal amino acid motif(S/TXV) that is recognized by PSD-95, shows a diffuse localization(Non-Patent Reference 7). From the foregoing, it is thought that theMAGUK protein is directly involved in the cross-linking and accumulationof cell membrane proteins through interaction via the PDZ domain.

In addition, activation of PKA (Protein Kinase A) in fibroblasts intowhich Kir has been transfected, results in the phosphorylation of serineat position 440 (S440) of the amino acid sequence of Kir, dissociationof Kir from PSD-95, and the diminution of potassium ion conductivity.When S440 is replaced with alanine, such effect by PKA is not observed.These results show that binding of Kir to PSD-95 clusterizes thechannels, and furthermore renders channel conductivity susceptible toPKA.

PSD-95 is a postsynaptic density protein, which is known to be involvedin a number of functions such as embryonic development, neurogenesis,neurotransmission, signal transduction, and protein complex formation.In addition, transferase activity, kinase activity, activity as ananchor protein in coupling membrane proteins (such as receptors and ionchannels) to the cytoskeleton, and activity as an adhesin/agglutininthat participates in the aggregation and conjunction of cells, have beenreported (Non-Patent References 4 and 8). From the foregoing, PSD-95 isthought to be involved in the control of neurotransmitter release, andin the inhibition of cell proliferation and cancers (Non-PatentReferences 9, 10, and 11).

PSD-95 has three PDZ domains, one SH3 domain (Src homology region 3domain), and one GK domain (guanylate kinase domain) in the amino acidsequence thereof.

PSD-95 binds via the PDZ domain thereof to the 2B subunit, which is acomponent of the NMDA receptor present in the postsynaptic membrane(Non-Patent References 2, 12, 13, and 14). It is thus believed thatPSD-95 is a cytoskeletal protein involved in the signal transductionmediated by the receptor, or the like.

In addition, PSD95 has been reported to interact via the GK domainthereof with PSD95/SAP90-associated protein-3 (hereinafter abbreviatedas SAPAP-3), in a rat (Non-Patent Reference 15). Rat SAPAP-3 isexpressed specifically in nerve cells, and is present abundantly in thepostsynaptic density fraction. In this report, rat SAPAP-3 is thought tobe involved in the maintenance of postsynaptic density structures byassembling postsynaptic density components, such as PSD95/SAP90, in themembrane region.

Meanwhile, the NMDA receptor is one type of glutamate receptor, which isthought to form an ion channel in the cell membrane, since it possessesa higher order structure, such as second or third structure, common toligand-dependent ion channels. The ion channel is permeable to calciumions and has the physiological function of increasing the calciumconcentration inside the postsynaptic membrane. The NMDA receptor isnormally obstructed by magnesium ions present in the synaptic cleft andis thought not to be involved in a single synapse transmission. However,when the membrane potential is depolarized as a result of frequentstimulation or the like, obstruction by the magnesium ions is removed,and the NMDA receptor is activated. Glutamic acid and aspartic acid canbe exemplified as in vivo ligands. Since memory impairment is provokedby administering amino-phosphono-valeric acid or MK-801 and the like(which are known as inhibitors of the NMDA receptor) directly into thebrain of a mammal, it is suggested that the NMDA receptor is involved inthe establishment of memory.

As described in the foregoing, the PDZ domain is anticipated to functionin a considerably wide range of biological phenomena. Therefore, byelucidating and controlling the complexes formed by proteins thatpossess the PDZ domain and the proteins involved in these complexes, itbecomes possible to prevent, treat, and diagnose diseases caused byanomalies in protein-protein interactions, such as the formation ofcytoskeletal structures, accumulation of membrane proteins, signaltransduction, formation of complexes, and maintenance of cell polarity.For example, by discovering a novel PSD that possesses the PDZ domain,it becomes possible to elucidate diseases in which an anomaly of the PSDis involved, for example, neurodegenerative diseases, as well as theprevention, treatment, and diagnosis thereof. That is to say, there is aneed for finding a protein that possesses the PDZ domain and a proteinthat interacts with that protein, as well as a method for controllingthe interactions of the two proteins, in the field of study ofprotein-protein interaction and recognition mechanism through the PDZdomain, as well as in the field of pharmaceutical development.

The references cited in the description of the present technicalbackground are listed below.

Non-Patent Reference 1: Saibokogaku, 2001, Vol. 20, pp. 1345-1349.

Non-Patent Reference 2: Niethammer, M., et al., Journal of Neuroscience,1996, Vol. 16, pp. 2157-2163.

Non-Patent Reference 3: Kornau, H. C., et al., Science, 1995, Vol. 269,pp. 1737-1740.

Non-Patent Reference 4: Kim, E., et al., Nature, 1995, Vol. 378, pp.85-88.

Non-Patent Reference 5: Kim, E., et al., Neuron, 1996, Vol. 17, pp.103-113.

Non-Patent Reference 6: Miller, B. M., et al., Journal of Neuroscience,1995, Vol. 15, pp. 2354-2366.

Non-Patent Reference 8: Stathakis, D., et al., Genomics, 1997, Vol. 44,pp. 71-82.

Non-Patent Reference 9: Hanada, T., et al., Journal of BiologicalChemistry, 2000, Vol. 275, pp. 28774-28784.

Non-Patent Reference 10: Migaud, M., et al., Nature, 1998, Vol. 396, pp.433-439.

Non-Patent Reference 11: Sattler, R., et al., Science, 1999, Vol. 284,pp. 1845-1848.

Non-Patent Reference 12: Monyer, H., et al., Science, 1992, Vol. 256,pp. 1217-1221.

Non-Patent Reference 13: Ishii, T., et al., Journal of BiologicalChemistry, 1993, Vol. 268, pp. 2836-2843.

Non-Patent Reference 14: Sheng, M., et al., Nature, 1994, Vol. 368, pp.144-147.

Non-Patent Reference 15: Takeuchi, M., et al., Journal of BiologicalChemistry, 1997, Vol. 272, pp. 11943-11951.

DISCLOSURE OF THE INVENTION

The present invention was achieved by way of discovering and using aprotein A that comprises a specific amino acid sequence having a PDZdomain acting as a module for carrying out protein-protein interactionsand is capable of forming a complex with an NMDA receptor; a protein Bthat interacts with the protein A; and genes coding therefor.

That is to say, one aspect of the present invention is an agent forcontrolling NMDA receptor mediated signal transduction, wherein theagent inhibits or promotes the binding of a polypeptide having the aminoacid sequence set forth in SEQ ID NO: 1 in the Sequence Listing, to theNMDA receptor, and/or, inhibits or promotes the interaction of apolypeptide having the amino acid sequence set forth in SEQ ID NO: 3 inthe Sequence Listing, with the polypeptide having the amino acidsequence set forth in SEQ ID NO: 1 in the Sequence Listing.

In addition, one aspect of the present invention is an agent forinhibiting NMDA receptor mediated signal transduction, wherein the agentinhibits binding of the polypeptide having the amino acid sequence setforth in SEQ ID NO: 1 in the Sequence Listing, to the NMDA receptor,and/or inhibits the interaction of the polypeptide having the amino acidsequence set forth in SEQ ID NO: 3 in the Sequence Listing, with thepolypeptide having the amino acid sequence set forth in SEQ ID NO: 1 inthe Sequence Listing.

In addition, one aspect of the present invention is an agent forenhancing NMDA receptor mediated signal transduction, wherein the agentpromotes the binding of the polypeptide having the amino acid sequenceset forth in SEQ ID NO: 1 in the Sequence Listing, to the NMDA receptor,and/or promotes the interaction of the polypeptide having the amino acidsequence set forth in SEQ ID NO: 3 in the Sequence Listing with thepolypeptide having the amino acid sequence set forth in SEQ ID NO: 1 inthe Sequence Listing.

In addition, one further aspect of the present invention is a method forcontrolling NMDA receptor mediated signal transduction, wherein themethod includes inhibiting or promoting the binding of the polypeptidehaving the amino acid sequence set forth in SEQ ID NO: 1 in the SequenceListing, to the NMDA receptor, and/or, inhibiting or promoting theinteraction of the polypeptide having the amino acid sequence set forthin SEQ ID NO: 3 in the Sequence Listing, with the polypeptide having theamino acid sequence set forth in SEQ ID NO: 1 in the Sequence Listing.

In addition, one further aspect of the present invention is a method forinhibiting NMDA receptor mediated signal transduction, wherein themethod includes inhibiting the binding of the polypeptide having theamino acid sequence set forth in SEQ ID NO: 1 in the Sequence Listing,to the NMDA receptor, and/or inhibiting the interaction of thepolypeptide having the amino acid sequence set forth in SEQ ID NO: 3 inthe Sequence Listing, with the polypeptide having the amino acidsequence set forth in SEQ ID NO: 1 in the Sequence Listing.

In addition, one aspect of the present invention is a method forpromoting NMDA receptor mediated signal transduction, wherein the methodincludes promoting the binding of the polypeptide having the amino acidsequence set forth in SEQ ID NO: 1 in the Sequence Listing, to the NMDAreceptor, and/or promoting the interaction of the polypeptide having theamino acid sequence set forth in SEQ ID NO: 3 in the Sequence Listing,with the polypeptide having the amino acid sequence set forth in SEQ IDNO: 1 in the Sequence Listing.

In addition, one aspect of the present invention is a polypeptideselected from the following polypeptides:

-   -   i. a polypeptide having the amino acid sequence set forth in SEQ        ID NO: 1 or 2 in the Sequence Listing;    -   ii. a polypeptide containing the polypeptide set forth above in        (i);    -   iii. a polypeptide having at least approximately 70% homology to        the polypeptide set forth above in (i) at the amino acid        sequence level and binding to the NMDA receptor/2B subunit; or    -   iv. a polypeptide having a mutation, which is a deletion, a        substitution, an addition, or an insertion of one to several        amino acids in the amino acid sequence of the polypeptide set        forth above in (i), and being capable of binding to the NMDA        receptor/2B subunit.

In addition, one further aspect of the present invention is apolypeptide selected from the following polypeptides, wherein thepolypeptide binds to an NMDA receptor/2B subunit:

-   -   i. a polypeptide having the amino acid sequence set forth in SEQ        ID NO: 1 or 2 in the Sequence Listing;    -   ii. a polypeptide containing the polypeptide set forth above in        (i);    -   iii. a polypeptide having at least approximately 70% homology to        the polypeptide set forth above in (i) at the amino acid        sequence level; or    -   iv. a polypeptide having a mutation, which is a deletion, a        substitution, an addition, or an insertion of one to several        amino acids in the amino acid sequence of the polypeptide set        forth above in (i).

In addition, one further aspect of the present invention is apolypeptide selected from the following polypeptides:

-   -   i. a polypeptide having the amino acid sequence set forth in SEQ        ID NO: 3 in the Sequence Listing;    -   ii. a polypeptide containing the polypeptide set forth above in        (i);    -   iii. a polypeptide having at least approximately 70% homology to        the polypeptide set forth above in (i) at the amino acid        sequence level, and interacting with the polypeptide having the        amino acid sequence set forth in SEQ ID NO: 1 in the Sequence        Listing; or    -   iv. a polypeptide having a mutation, which is a deletion, a        substitution, an addition, or an insertion of one to several        amino acids in the amino acid sequence of the polypeptide set        forth above in (i) and interacting with the polypeptide having        the amino acid sequence set forth in SEQ ID NO: 1 in the        Sequence Listing.

In addition, one aspect of the present invention is a polypeptideselected from the following polypeptides, wherein the polypeptideinteracts with the polypeptide having the amino acid sequence set forthin SEQ ID NO: 1 in the Sequence Listing:

-   -   i. a polypeptide having the amino acid sequence set forth in SEQ        ID NO:, 3 in the Sequence Listing;    -   ii. a polypeptide containing the polypeptide set forth above in        (i);    -   iii. a polypeptide having at least approximately 70% homology to        the polypeptide set forth above in (i) at the amino acid        sequence level; or    -   iv. a polypeptide having a mutation, which is a deletion, a        substitution, an addition, or an insertion of one to several        amino acids, in the amino acid sequence, of the polypeptide set        forth above in (i).

In addition, one aspect of the present invention is a polypeptideselected from the following polypeptides, wherein the polypeptideamplifies NMDA receptor mediated signal transduction, in the presence ofthe polypeptide having the amino acid sequence set forth in SEQ ID NO: 1in the Sequence Listing:

-   -   i. a polypeptide having the amino acid sequence set forth in SEQ        ID NO: 3 in the Sequence Listing;    -   ii. a polypeptide containing the polypeptide set forth above in        (i);    -   iii. a polypeptide having at least approximately 70% homology to        the polypeptide set forth above in (i) at the amino acid        sequence level; or    -   iv. a polypeptide having a mutation, which is a deletion, a        substitution, an addition, or an insertion of one to several        amino acids in the amino acid sequence of the polypeptide set        forth above in i).

In addition, one aspect of the present invention is one of the followingpolypeptides, which binds to the NMDA receptor/2B subunit but does notinteract with the polypeptide having the amino acid sequence set forthin SEQ ID NO: 3 in the Sequence Listing:

-   -   i. a polypeptide having at least approximately 70% homology at        the amino acid sequence level to the polypeptide having the        amino acid sequence set forth in SEQ ID NO: 1 or 2 in the        Sequence Listing; or    -   ii. a polypeptide having a mutation, which is a deletion, a        substitution, an addition, or an insertion of one to several        amino acids, in the amino acid sequence of the polypeptide set        forth above in (i).

In addition, one further aspect of the present invention is a peptidecomprising at least five consecutive amino acid residues from the aminoacid sequence set forth in SEQ ID NO: 1 or 2 in the Sequence Listing.

In addition, one aspect of the present invention is a peptide comprisingat least five consecutive amino acid residues within the amino acidsequence set forth in SEQ ID NO: 1 or 2 in the Sequence Listing andbinding to the NMDA receptor.

In addition, one aspect of the present invention is a peptide comprisingat least five consecutive amino acid residues within the amino acidsequence set forth in SEQ ID NO: 1 in the Sequence Listing, andinteracting with the polypeptide having the amino acid sequence setforth in SEQ ID NO: 3 in the Sequence Listing.

In addition, one further aspect of the present invention is a peptidecomprising at least five consecutive amino acid residues within theamino acid sequence set forth in SEQ ID NO: 3 in the Sequence Listing.

In addition, one aspect of the present invention is a peptide comprisingat least five consecutive amino acid residues within the amino acidsequence set forth in SEQ ID NO: 3 in the Sequence Listing, andinteracting with the polypeptide having the amino acid sequence setforth in SEQ ID NO: 1 in the Sequence Listing.

In addition, one aspect of the present invention is an agent forcontrolling NMDA receptor mediated signal transduction, wherein theagent includes an effective amount of at least one kind of polypeptideor peptide selected from the aforementioned polypeptides and peptides.

In addition, one aspect of the present invention is an agent forinhibiting NMDA receptor mediated signal transduction, wherein the agentincludes an effective amount of at least one kind of polypeptide orpeptide selected from the aforementioned polypeptides and peptides.

In addition, one aspect of the present invention is an agent forpromoting NMDA receptor mediated signal transduction, wherein the agentincludes an effective amount of at least one type of polypeptide orpeptide selected from the aforementioned polypeptides and peptides.

In addition, one aspect of the present invention is a method forcontrolling NMDA receptor mediated signal transduction, wherein themethod includes using at least one kind of polypeptide or peptideselected from the aforementioned polypeptides and peptides.

In addition, one aspect of the present invention is a method forinhibiting NMDA receptor mediated signal transduction, wherein themethod includes using at least one kind of polypeptide or peptideselected from the aforementioned polypeptides and peptides.

In addition, one aspect of the present invention is a method forpromoting NMDA receptor mediated signal transduction, wherein the methodincludes using at least one kind of polypeptide or peptide selected fromthe aforementioned polypeptides and peptides.

In addition, one further aspect of the present invention is apolynucleotide including a nucleotide sequence coding for any of theaforementioned polypeptides or peptides, or a complementary nucleotidesequence thereof.

In addition, one further aspect of the present invention is apolynucleotide having a nucleotide sequence set forth in SEQ ID NO: 4 or5 in the Sequence Listing, or a complementary nucleotide sequencethereof.

In addition, one aspect of the present invention is a polynucleotidehaving a nucleotide sequence set forth in SEQ ID NO: 6 in the SequenceListing, or a complementary nucleotide sequence thereof.

In addition, one further aspect of the present invention is apolynucleotide that hybridizes with the aforementioned polynucleotideunder stringent conditions.

Furthermore, one aspect of the present invention is a recombinant vectorthat contains the aforementioned polynucleotide.

Furthermore, one aspect of the present invention is the aforementionedrecombinant vector, wherein the recombinant vector is a recombinantexpression vector.

In addition, one aspect of the present invention is a transformant thathas been transfected with the aforementioned recombinant vector.

In addition, one aspect of the present invention is a method formanufacturing the aforementioned polypeptide or peptide, wherein themethod includes a step of culturing the transformant that has beentransformed with the aforementioned recombinant vector, or a cell-freeprotein synthesis means that uses the recombinant vector.

Furthermore, one aspect of the present invention is an antibody thatimmunologically recognizes the aforementioned polypeptide and/orpeptide.

Furthermore, one additional aspect of the present invention is theaforementioned antibody that inhibits the function of the aforementionedpolypeptide.

In addition, one aspect of the present invention is the aforementionedantibody which inhibits the interaction of the polypeptide having theamino acid sequence set forth in SEQ ID NO: 1 in the Sequence Listing,with the polypeptide having the amino acid sequence set forth in SEQ IDNO: 3 in the Sequence Listing.

In addition, one further aspect of the present invention is a method foridentifying a compound that interacts with the aforementionedpolypeptide and inhibits or promotes the function thereof and/or acompound that interacts with the aforementioned polynucleotide andinhibits or promotes the expression thereof, wherein the method includesusing at least one selected from the group consisting of theaforementioned polypeptides, polynucleotides, recombinant vectors,transformants, and antibodies.

In addition, one further aspect of the present invention is a method foridentifying a compound that interacts with the aforementionedpolypeptide and inhibits or promotes the function thereof, and/or acompound that interacts with the aforementioned polynucleotide andinhibits or promotes the expression thereof, wherein the method includescontacting the compound with the polypeptide or the polynucleotide underconditions where the interaction of the compound with the polypeptide orthe interaction of the compound with the polynucleotide are allowed, anddetermining whether the compound interacts with the polypeptide or thepolynucleotide and inhibits or promotes the function of the polypeptideor the expression of the polynucleotide by detecting presence, absence,or variation of a signal which results from the interaction of thecompound with the polypeptide or the interaction of the compound withthe polynucleotide.

In addition, one further aspect of the present invention is a method foridentifying a compound that interacts with the aforementionedpolypeptides and inhibits or promotes the function thereof, and/or acompound that interacts with the polynucleotides and inhibits orpromotes the expression thereof, wherein the method includes contactingthe compound with the transformant, and determining whether the compoundinhibits or promotes the expression or function of the polypeptide byusing a system that uses a signal and/or a marker being capable ofdetecting presence or absence of the expression or the function of thepolypeptide to detect presence, absence, or variation of the signaland/or marker.

In addition, one further aspect of the present invention is a method foridentifying a compound that inhibits or promotes the binding of thepolypeptide having the amino acid sequence set forth in SEQ ID NO: 1 inthe Sequence Listing, to the NMDA receptor, wherein the method includesusing at least one selected from the group consisting of theaforementioned polypeptides, polynucleotides, recombinant vectors,transformants, and antibodies.

In addition, one further aspect of the present invention is a method foridentifying a compound that inhibits or promotes the interaction of thepolypeptide having the amino acid sequence set forth in SEQ ID NO: 1 inthe Sequence Listing, with the polypeptide having the amino acidsequence set forth in SEQ ID NO: 3 in the Sequence Listing, wherein themethod includes using at least one selected from the group consisting ofthe aforementioned polypeptides, polynucleotides, recombinant vectors,transformants, and antibodies.

In addition, one aspect of the present invention is a compound that hasbeen identified by any of the aforementioned methods.

Furthermore, one additional aspect of the present invention is acompound that interacts with the aforementioned polypeptide and inhibitsor promotes the function thereof.

In addition, one aspect of the present invention is a compound thatinteracts with the aforementioned polypeptide and inhibits or promotesthe interaction of the polypeptide with the polypeptide having the aminoacid sequence set forth in SEQ ID NO: 1 in the Sequence Listing.

In addition, one aspect of the present invention is a compound thatinteracts with the aforementioned polypeptide and inhibits or promotesthe amplification of the NMDA receptor mediated signal transduction inthe presence of the polypeptide and the polypeptide having the aminoacid sequence set forth in SEQ ID NO: 1 in the Sequence Listing.

Furthermore, one additional aspect of the present invention is acompound that interacts with the aforementioned polynucleotide andinhibits or promotes the expression thereof.

In addition, one further aspect of the present invention is apharmaceutical composition comprising an effective dose of at least oneselected from the group consisting of the aforementioned polypeptides,peptides, polynucleotides, recombinant vectors, transformants,antibodies, compounds, controlling agents, inhibiting agents, andpromoting agents.

In addition, one further aspect of the present invention is an agent forpreventing, treating, or improving a disease caused by an anomaly in theNMDA receptor mediated signal transduction, wherein the agent includesan effective dose of at least one selected from the group consisting ofthe aforementioned polypeptides, peptides, polynucleotides, recombinantvectors, transformants, antibodies, compounds, controlling agents,inhibiting agents, and promoting agents.

In addition, one aspect of the present invention is an agent forpreventing, treating, or improving a disease caused by an anomaly inmemory recall, wherein the agent includes an effective dose of at leastone selected from the group consisting of the aforementionedpolypeptides, peptides, polynucleotides, recombinant vectors,transformants, antibodies, compounds, controlling agents, inhibitingagents, and promoting agents.

In addition, one aspect of the present invention is an agent forpreventing, treating, or improving a neurodegenerative disease, whereinthe agent includes an effective dose of at least one selected from thegroup consisting of the aforementioned polypeptides, peptides,polynucleotides, recombinant vectors, transformants, antibodies,compounds, controlling agents, inhibiting agents, and promoting agents.

In addition, one aspect of the present invention is an agent forpreventing, treating, or improving Alzheimer's disease, wherein theagent includes an effective dose of at least one selected from the groupconsisting of the aforementioned polypeptides, peptides,polynucleotides, recombinant vectors, transformants, antibodies,compounds, controlling agents, inhibiting agents, and promoting agents.

In addition, one further aspect of the present invention is a method forpreventing, treating, or improving a disease caused by an anomaly in theNMDA receptor mediated signal transduction, wherein the method includesadministering at least one selected from the group consisting of theaforementioned polypeptides, peptides, polynucleotides, recombinantvectors, transformants, antibodies, compounds, controlling agents,inhibiting agents, and promoting agents.

In addition, one further aspect of the present invention is a method forpreventing, treating or improving a disease caused by an anomaly inmemory recall, wherein the method includes administering at least oneselected from the group consisting of the aforementioned polypeptides,peptides, polynucleotides, recombinant vectors, transformants,antibodies, compounds, controlling agents, inhibiting agents, andpromoting agents.

In addition, one further aspect of the present invention is a method forpreventing, treating, or improving a neurodegenerative disease, whereinthe method includes administering at least one selected from the groupconsisting of the aforementioned polypeptides, peptides,polynucleotides, recombinant vectors, transformants, antibodies,compounds, controlling agents, inhibiting agents, and promoting agents.

In addition, one further aspect of the present invention is a method forpreventing, treating, or improving Alzheimer's disease, wherein themethod includes administering at least one selected from the groupconsisting of the aforementioned polypeptides, peptides,polynucleotides, recombinant vectors, transformants, antibodies,compounds, controlling agents, inhibiting agents, and promoting agents.

Furthermore, one additional aspect of the present invention is a methodfor quantitatively or qualitatively measuring the aforementionedpolypeptides or polynucleotides.

Furthermore, one aspect of the present invention is a reagent kitcontaining at least one selected from the group consisting of theaforementioned polypeptides, peptides, polynucleotides, recombinantvectors, transformants, and antibodies.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic drawing comparing the characteristics of the aminoacid sequences of a novel PSD (referred to as Protein-X) and PSD-95.

FIG. 2 is an electropherogram obtained when a 6× His-tag fusion proteinof the novel PSD (Protein-X) was expressed in Escherichia coli culturedat 37° C.

FIG. 3 is an electropherogram of the 6× His-tag fusion protein of thenovel PSD (Protein-X), showing that the amount expressed in the solublefraction of Escherichia coli cultured at 37° C. is low.

FIG. 4 is an electropherogram showing that the 6× His-tag fusion proteinof the novel PSD (Protein-X) is expressed in the soluble fraction ofEscherichia coli cultured at 25° C.

FIG. 5 shows a result of Western-blotting, demonstrating the expressionand the expressed amount of the 6× His-tag fusion protein of the novelPSD (Protein-X) or of a PDZ domain deletion mutant thereof.

FIG. 6 shows a result of Western-blotting, demonstrating the expressionof a GST fusion protein of an NMDA receptor/2B subunit.

FIG. 7 shows a result of Western-blotting, demonstrating the expressionand the expressed amount of the GST fusion protein of the NMDAreceptor/2B subunit as well as the expression and the expressed amountof GST.

FIG. 8 is a result obtained by the Overlay method, showing that thenovel PSD (Protein-X) binds to a NMDA receptor/2B subunit, whereas thePDZ domain deletion mutant of the novel PSD (Protein-X) does not.

FIG. 9 shows that the novel PSD (Protein-X) and the PDZ domain deletionmutant thereof do not bind to GST.

FIG. 10 is an electropherogram obtained when the 6× His-tag fusionprotein of the PDZ domain deletion mutant of the novel PSD (Protein-X)was expressed in Escherichia coli cultured at 37° C.

FIG. 11 is an electropherogram showing that the 6× His-tag fusionprotein of the PDZ domain deletion mutant of the novel PSD (Protein-X)is expressed in the soluble fraction of Escherichia coli cultured at 37°C.

FIG. 12 is an electropherogram showing the expression of the GST fusionprotein of the NMDA receptor/2B subunit.

FIG. 13 is a restriction map of a vector carrying a novel PSD(Protein-X) gene.

FIG. 14 is a restriction map of a vector carrying an NMDA receptor/2Bsubunit gene.

FIG. 15 is a restriction map of a vector carrying an NMDA receptor Igene.

FIG. 16 is a restriction map of a vector carrying a PSD-95 gene.

FIG. 17 shows that an electrical response generated by an NMDA receptorstimulation is enhanced by the novel PSD (Protein-X).

FIG. 18A is an electropherogram showing that the induction of theexpression of a His-tag fused human PJ01087 protein (can be referred toas Protein-Y) in Escherichia coli was demonstrated by Western-blottingusing an anti-His-tag antibody. In the figure, the arrow indicates thehuman PJ01087 protein (Protein-Y).

FIG. 18B is an electropherogram showing that the induction of theexpression of a GST fused human PJ01087 protein (can be referred to asProtein-Y) in Escherichia coli, was demonstrated by Western-blottingusing an anti-GST antibody. In the figure, the arrow indicates the humanPJ01087 protein (Protein-Y).

FIG. 19 is a restriction map of a vector carrying a human PJ01087 gene(SEQ ID NO: 6 in the Sequence Listing).

FIG. 20 shows that the human PJ01087 protein (Protein-Y) furtherintensifies the enhancement effect by the novel PSD (Protein-X) in theelectrical response generated by the NMDA receptor stimulation, but doesnot intensify the enhancement effect by PSD-95.

FIG. 21 shows that the enhancement, by the novel PSD (Protein-X), of themagnitude of variation in the electrical current generated by the NMDAreceptor stimulation is further intensified by the human PJ01087 protein(Protein-Y).

FIG. 22 shows a result obtained by mixing the His-tag fused humanPJ01087 protein (Protein-Y) with the GST fused novel PSD (Protein-X)(hj02537), followed by immunoprecipitating them with the anti-GSTantibody, and finally detecting the immunoprecipitated proteins with theanti-GST antibody.

FIG. 23 shows a result obtained by mixing the His-tag fused humanPJ01087 protein (Protein-Y) and the GST fused novel PSD (Protein-X)(hj02537), followed by immunoprecipitating them with the anti-GSTantibody, and finally detecting the immunoprecipitated proteins with theanti-His-tag antibody.

DETAILED DESCRIPTION OF THE INVENTION

The present invention claims priority from Japanese Patent ApplicationNumbers 2001-354678, 2002-46786, and 2002-229863, which are incorporatedherein by reference.

Technical and scientific terms used in the present specification, unlessseparately defined, have meanings that are normally understood by thoseskilled in the art to which the present invention belongs. In thepresent specification, reference is made to a variety of methods knownto those skilled in the art. Data from publications and the like thatdisclose such cited well-known methods are completely incorporatedherein, in their entirety, by reference.

Hereinafter, for the present invention, a mode of embodiment of theinvention is described in more detail. The following detaileddescription is illustrative and merely explanatory, and does not limitthe present invention in any way.

Novel PSD and Protein Interacting with the PSD

The present invention provides a novel human PSD and a proteininteracting with the PSD, as well as the genes coding for each of these.

The novel human PSD is a protein coded by clone hj02537, which wasselected from a cDNA library (constructed by human brain mRNA andcovered relatively long length DNA), as a cloned gene that has a PDZdomain, a SH3 domain and a GK domain. This protein was obtained asexpressed product of Escherichia coli that was transformed with anexpression plasmid carrying the hj02537 gene.

Hereinafter, the human PSD of the present invention may be referred toas Protein-X, and the gene coding for the PSD may be referred to as theProtein-X gene. The Protein-X gene comprises 4941 bps (SEQ ID NO: 4 inthe Sequence Listing), which contains the full length of an open readingframe (ORF) including 1731 bps; the gene product thereof comprising 576amino acid residues (SEQ ID NO: 1 in the Sequence Listing). Similarly tothe known PSD, the Protein-X possesses a PDZ domain from isoleucine(lIe) 139 to glycine (Gly) 219, an SH3 domain from methionine (Met) 231to arginine (Arg) 296, and a GK domain from threonine (Thr) 404 toasparagine (Asn) 500, in the amino acid sequence thereof. From theforegoing, Protein-X was inferred to be a homolog of DLG-like PDZproteins that include PSD-95 (refer to FIG. 1). Therefore, Protein-X isthought to have similar activities to PSD-95, so that it is anticipatedto exist in the vicinity of membrane receptors and ion channels to forman apparatus (which means a functional structure); to participate in thecontrol of neurotransmitter release and signal transfer between cells.

Indeed, similarly to PSD-95, Protein-X can bind to an NMDA receptor/2Bsubunit via the PDZ domain; and furthermore, it can exhibit anamplification activity greater than that of PSD-95, in response to asignal resulting from the stimulation of the NMDA receptor by a ligand.That is to say, Protein-X is thought to form a complex with an NMDAreceptor and enhance the NMDA receptor mediated signal transduction.However, Protein-X and PSD-95 carry different numbers of PDZ domains:three in PSD-95, and one in Protein-X. Thus, there is a possibility thatProtein-X carries a receptor signal different from that of PSD-95.Alternately, Protein-X can probably support for PSD-95-like signaltransduction or exhibit binding selectivity to other receptors and thelike due to the number of PDZ domains.

In addition, the present invention provides a polypeptide in which the85 amino acids on the N-terminal side of Protein-X have been deleted(SEQ ID NO: 2 in the Sequence Listing). This polypeptide is encoded by agene clone pj02677 obtained from a cDNA library (constructed by humanbrain hippocampus mRNA and covered relatively long length DNA). Thepj02677 is a polynucleotide comprising 4370 bases (SEQ ID NO: 5 in theSequence Listing), which codes for a polypeptide comprising 491 aminoacids (SEQ ID NO: 2 in the Sequence Listing). The amino acid sequence ofthe polypeptide encoded by the pj02677 is identical to that of Protein-Xwith the exception of the deletion of the 85 amino acid residues on theN-terminal side. Therefore, it possesses one PDZ domain, one SH3 domain,and one GK domain in the amino acid sequence thereof; this fact indicatethat it may bind to a NMDA receptor/2B subunit in a similar manner asProtein-X does and may exhibit a similar biological activity as that ofProtein-X. The expression of pj02677 was observed extensively in variousorgans such as the brain (including cerebellum and the like), the heart,the liver, the skeletal muscle, the kidneys, the pancreas, the spleen,and the spinal cord. As pj02677 is believed to result from theaforementioned hj02537 with a deletion of the nucleotide sequence fromthe 5′ end side thereof, the Protein-X gene is also probably expressedin these organs.

Meanwhile, the protein that interacts with Protein-X is encoded by clonePJ01087, which was extracted from the cDNA analysis information database(containing information of cDNA derived from human brain almostcompletely mRNA) of the Kazusa DNA Research Institute, as a gene havinghomology of 96% with the rat PSD95/SAP90-associated protein 3 (SAPAP-3)(GenBank: Accession No: U67139). This protein was obtained as expressedproduct of Escherichia coli which was transformed with a plasmidcarrying the PJ01087 gene. Hereinafter, this protein is referred to asthe PJ01087 protein, and the gene that codes for the protein is referredto as the PJ01087 gene. In addition, the PJ01087 protein can be referredto as Protein-Y. The PJ01087 gene comprises 3705 bps (SEQ ID NO: 6 inthe Sequence Listing), which contains the full length of the ORFcomprising 2940 bps; the gene product thereof comprises 979 amino acidresidues (SEQ ID NO: 3 in the Sequence Listing). In addition, it wasrevealed that Protein-Y is expressed specifically in the brain, byreverse transcription polymerase chain reaction (RT-PCR) andenzyme-linked immunosorbent assay (ELISA).

Protein-Y markedly amplified the signal from the NMDA receptor in thepresence of Protein-X. The signal amplification caused by Protein-Y andProtein-X was considerably greater than the amplification caused byProtein-X alone. On the other hand, the signal amplification byProtein-Y was not observed in the presence of PSD-95. From theforegoing, it was revealed that Protein-Y interacts with Protein-X, andthat the signal transduction resulting from the stimulation of the NMDAreceptor by a ligand is markedly amplified by the interaction.

In the present specification, Protein-Y interacting with Protein-X meansthat both proteins mutually exert an activity in a certain scheme,resulting in enhancement of the function of each protein. The functionof each protein can be exemplified by the promotion of physiologicalfunctions of membrane receptors and ion channels (such as the promotionof the stabilization of the membrane receptors and the acceleration ofreceptor mediated signal transduction). The membrane receptor and theion channel can be exemplified by NMDA receptor. The interaction schemescan be exemplified by binding, temporary binding, or the formation of acomplex via another substance: however it is not limited thereto. Sincethe binding of Protein-Y to Protein-X was not observed inimmunoprecipitation analyses, the interaction between the two proteinsmay not be accomplished through binding, but through some alternativescheme.

These findings suggest that a signal of a membrane receptor, forexample, a signal of an NMDA receptor, can be amplified by theoverexpression of Protein-X or by promoting the biological activitythereof (for example, the interaction with another protein or aguanylate kinase activity). In addition, it is suggested that anabnormal signal from a membrane receptor, for example, an abnormalsignal from of an NMDA receptor, can be normalized by inhibiting theexpression of Protein-X or by inhibiting the biological activity thereof(for example, the interaction with another protein or a guanylate kinaseactivity). Similarly, a signal from a membrane receptor can be amplifiedby the overexpression of Protein-Y or by promoting the biologicalactivity thereof. In addition, an abnormal signal of a membrane receptorcan be normalized by inhibiting the expression of Protein-Y or byinhibiting the biological activity thereof. That is to say, the signaltransduction, and the like, of a membrane receptor can be controlled byinhibiting or promoting the interaction of Protein-X with the membranereceptor or with a protein that participates in the receptor mediatedsignal transduction. Herein, “control” means “inhibition” or“promotion.” In addition, “promotion” includes -the common meanings of“enhancement” and “amplification.” For instance, the NMDA receptormediated signal transduction and the like can be controlled byinhibiting or promoting the interaction of Protein-X with the NMDAreceptor, or inhibiting or promoting the interaction of Protein-X withProtein-Y. Therefore, Protein-X and Protein-Y, as well as an inhibitorand a promoting agent for the expression thereof or for the biologicalactivity thereof, can be applied to the prevention, improvement, ortreatment of a disease: caused by an anomaly in Protein-X; caused by ananomaly in Protein-Y; caused by an anomaly in both proteins; or causedby an anomaly in the interaction between the two proteins. That is tosay, they can be used for a prophylactic agent, an improving agent, or atherapeutic agent of these diseases, as well as in a prevention method,an improvement method, or a treatment method. A disease based on ananomaly of a membrane receptor mediated signal transduction (whereProtein-X and/or Protein-Y is implicated) can be cited as such adisease. More specifically, it can be exemplified by neurodegenerativediseases such as Alzheimer's disease, Parkinson's disease, andpolyglutamine disease; however it is not limited thereto.

Polypeptides or Peptides

In the present specification, a longer chain peptide (having any numberof peptides containing two or more amino acids that bind to one anotherthrough a peptide bond or a modified peptide bond), such as a protein,is called a polypeptide; a shorter chain peptide (sometimes alsoreferred to as oligopeptide or oligomer) is simply called a peptide. Inthe present specification, an amino acid can sometimes be represented bythree letters.

The polypeptide according to the present invention is a gene product ofthe Protein-X gene; it can be obtained by expressing the gene in a cellsuch as Escherichia coli. In addition, it can be a polypeptide obtainedby chemical synthesis based on the amino acid sequence of thepolypeptide of interest; it can also be a polypeptide derived from cellsor from any tissue where these cells are exist. The polypeptide can be apolypeptide comprising an amino acid sequence that is identical orsubstantially identical to the amino acid sequence set forth in SEQ IDNO: 1 or 2 in the Sequence Listing. As the amino acid sequence that issubstantially identical to the amino acid sequences set forth in SEQ IDNO: 1 or 2 in the Sequence Listing, it is not limited as long as thepolypeptide can at least bind to an NMDA receptor/2B subunit, and can,for example be a polypeptide that contains a polypeptide having theamino acid sequences set forth in SEQ ID NO: 1 or 2 in the SequenceListing. Alternatively, it can be a polypeptide that has an amino acidsequence homologus to the polypeptide having the amino acid sequencesset forth in SEQ ID NO: 1 or 2 in the Sequence Listing, with 70% orgreater, preferably 80% or greater, more preferably 90% or greater, andeven more preferably 95% or greater, and that is capable of binding toan NMDA receptor/2B subunit. A polypeptide further having a guanylatekinase activity is more preferred. In addition, a polypeptide possessingone PDZ domain is further preferable.

Binding to an NMDA receptor/2B subunit can be measured by any well-knownmethod such as the Overlay method, as described in the examples below.In addition, the guanylate kinase activity can be measured by anywell-known method. Techniques for determining the amino acid sequencehomology are well-known, for example, a method that directly determinesthe amino acid sequence, or a method that determines the nucleotidesequence of the cDNA and then infers the amino acid sequence encodedthereby. Needless to say, homologous gene products of animal speciesother than human are also included in the present invention. Inaddition, based on a polypeptide thus specified and using an indicator(for example, binding to the NMDA receptor/2B subunit and/or guanylatekinase activity), a polypeptide can be provided, which includes an aminoacid sequence having a mutation, which can be a deletion, asubstitution, an addition, or an insertion of one or more amino acids,for instance one to 100, preferably one to 30, more preferably one to20, even more preferably one to 10, and particularly preferably one toseveral amino acids. These polypeptides are also included in thepolypeptides comprising an amino acid sequence that is substantiallyidentical to the amino acid sequences set forth in SEQ ID NO: 1 or 2 inthe Sequence Listing. As such a polypeptide, for example, thepolypeptide having the amino acid sequence set forth in SEQ ID NO: 2 inthe Sequence Listing is a polypeptide comprising an amino acid sequencethat is substantially identical to the amino acid sequence set forth inSEQ ID NO: 1 in the Sequence Listing. The aforementioned polypeptideshaving a mutation can be those that exist naturally, or those into whicha mutation has been introduced. In addition, the present invention canalso include a polypeptide that comprises an amino acid sequencesubstantially identical to the amino acid sequence set forth in SEQ IDNO: 1 in the Sequence Listing, and does not interact with thepolypeptide having the amino acid sequence set forth in SEQ ID NO: 3 inthe Sequence Listing. Such a polypeptide is inferred to inhibit theinteraction of Protein-X with Protein-Y by competing with Protein-X.Thus, such a polypeptide probably inhibits the NMDA receptor mediatedsignal transduction.

Another polypeptide according to the present invention is a gene productof the Protein-Y gene; it can be obtained by expressing the gene in acell such as Escherichia coli. In addition, it can be a polypeptideobtained by chemical synthesis based on the amino acid sequence of thepolypeptide of interest; it can also be a polypeptide derived from cellsor from any tissue where these cells are present. The polypeptide can bea polypeptide comprising an amino acid sequence that is identical orsubstantially identical to the amino acid sequence set forth in SEQ IDNO: 3 in the Sequence Listing. As the amino acid sequence that issubstantially identical to the amino acid sequence set forth in SEQ IDNO: 3 in the Sequence Listing, it is not limited as long as thepolypeptide can at least interact with the polypeptide having the aminoacid sequence set forth in SEQ ID NO: 1 in the Sequence Listing, andcan, for instance be a polypeptide that contains a polypeptide havingthe amino acid sequence set forth in SEQ ID NO: 3 in the SequenceListing. Alternatively, it can be a polypeptide that has an amino acidsequence homologous to the polypeptide having the amino acid sequenceset forth in SEQ ID NO: 3 in the Sequence Listing, with 70% or greater,preferably 80% or greater, more preferably 90% or greater, and even morepreferably 95% or greater, and that is capable interacting with thepolypeptide having the amino acid sequence set forth in SEQ ID NO: 1 inthe Sequence Listing. In addition, it can also be a polypeptide thatamplifies NMDA receptor mediated signal transduction in the presence ofthe polypeptide having the amino acid sequence set forth in SEQ ID NO: 1in the Sequence Listing. In this case, the amino acid sequence ofSAPAP-3 is not included in the present invention.

In addition, in terms of the concept of a module (the structural unitdetermined by the tertiary structure of the protein, which often worksalso as a functional unit), a polypeptide comprising at least fiveconsecutive amino acids among the amino acid sequence set forth in SEQID NO: 3 in the Sequence Listing, can also be used for the interactingpeptide. The number of amino acid residues of the module is known to be15 on average; however considering its function of being capable ofworking as an antigen or the like, the five amino acid residues may forma sufficient functional unit. The interaction with the polypeptidehaving the amino acid sequence set forth in SEQ ID NO: 1 in the SequenceListing, can be assayed, for example, by co-expressing the polypeptide,a polypeptide that is the object of the assay, and the NMDA receptortogether in a cell, followed by measuring the intracellular signaltransduction resulting from the stimulation of the NMDA receptor by aligand; the mesurement can performed by using a well-known method (seeExample 4).

Techniques for determining the amino acid sequence homology arewell-known. For example, a method that directly determines the aminoacid sequence, or a method that determines the nucleotide sequence ofthe cDNA and then infers the amino acid sequence encoded thereby can beused. Based on a polypeptide thus specified and using an indicator (forexample, the interaction with the polypeptide having the amino acidsequence set forth in SEQ ID NO: 3 in the Sequence Listing; thepromotion of the NMDA receptor mediated signal transduction; theamplification of the NMDA receptor mediated signal transduction in thepresence of the polypeptide having the amino acid sequence set forth inSEQ ID NO: 1 in the Sequence Listing), a polypeptide can be provided.The polypeptide can include an amino acid sequence having a mutation,which can be a deletion, a substitution, an addition, or an insertion ofone or more amino acids, for example, one to 100, preferably one to 30,more preferably one to 20, even more preferably one to 10, andparticularly preferably one to several amino acids. These polypeptidesare also included in the polypeptides comprising an amino acid sequencethat is substantially identical to the amino acid sequence set forth inSEQ ID NO: 3 in the Sequence Listing. In this case, the amino acidsequence of SAPAP-3 is not included in the present invention. Thepeptides having a mutation can be those that exist naturally, or thoseinto which a mutation has been introduced.

Methods for introducing a mutation such as a deletion, a substitution,an addition, and/or an insertion are well-known. For instance,site-directed mutagenesis, homologous gene recombination, primerextension or polymerase chain reaction (PCR) can be used alone or inappropriate combination, according to the methods described in manualssuch as Sambrook et al. Eds., Molecular Cloning: A Laboratory Manual,2nd Ed., Cold Spring Harbor Press, 1989; Masami Muramatsu Ed.,Laboratory Manual Genetic Engineering, Maruzen Co. Ltd., 1988; Ehrlich,H. E. Ed., PCR Technology—Principles and Applications for DNAAmplification, Stockton Press, 1989. These methods may be modified foruse. For instance, Ulmer's technique (Science, 1983, Vol. 219, p. 666 etseq.) can be used. Insuring that the fundamental characteristics(physical properties, functions, biological activity, or immunologicalactivity and the like) of these polypeptides are not changed by theintroduction of such mutation(s), as reciprocal substitution among, forexample, homologous amino acids (polar amino acids, non-polar aminoacids, hydrophobic amino acids, hydrophilic amino acids, positivelycharged amino acids, negatively charged amino acids, aromatic aminoacids, and the like) is readily inferred. Furthermore, thesepolypeptides can be altered to the extent that no significant functionalalteration is involved, such as modifying their constituent amino groupor carboxyl group and the like.

The polypeptide comprising an amino acid sequence that is substantiallyidentical to the amino acid sequence set forth in SEQ ID NO: 1 in theSequence Listing, can be a polypeptide with a function or an activitythat is promoted or decreased compared to the polypeptide having theamino acid sequence set forth in SEQ ID NO: 1 in the Sequence Listing.For instance, the ability thereof to form a complex with an NMDAreceptor, the guanylate kinase activity thereof, and/or the extent ofthe interaction thereof with the polypeptide having the amino acidsequence set forth in SEQ ID NO: 3 in the Sequence Listing, can bedifferent. More specifically, for instance, the magnitude of thepromotion of the NMDA receptor mediated signal transduction can bedifferent, the function thereof can be additionally promoted, or thefunction thereof can be decreased.

The polypeptide comprising an amino acid sequence that is substantiallyidentical to the amino acid sequence set forth in SEQ ID NO: 3 in theSequence Listing, can be a polypeptide with a function or an activitythat is promoted or more decreased compared to the polypeptide havingthe amino acid sequence set forth in SEQ ID NO: 3 in the SequenceListing. For instance, the extent of the interaction thereof with thepolypeptide having the amino acid sequence set forth in SEQ ID NO: 1 inthe Sequence Listing, can be different, the interaction can be promoted,or the interaction can be decreased. More specifically, for instance,the magnitude of the promotion of the NMDA receptor mediated signaltransduction can be different, the function thereof can be promoted, orthe function thereof can be decreased.

The present invention also includes the polypeptides to which adifferent type of protein or substance has been ligated, for example, acarrier and the like. For instance, a different type of protein orpeptide (for example, alkaline phosphatase, β-galactosidase, glutathioneS-transferase (GST), or an Fc fragment of an immunoglobulin such asimmunoglobulin G (IgG)) can be ligated to the N-terminus or theC-terminus thereof, directly or indirectly via a linker peptide, byusing well-known methods such as gene engineering techniques, in orderto facilitate the detection or purification of the polypeptide and thelike of the present invention, or in order to add a different function.

In addition, the present invention includes a polypeptide or a peptidethat has a partial sequence of the polypeptide having the amino acidsequence set forth in SEQ ID NO: 1 or 3 in the Sequence Listing. Thepolypeptide or the peptide that has the partial sequence comprisesconsecutive amino acids of 5 or more, preferably 8 or more, morepreferably 12 or more, further preferably 15 amino acids or more, as theminimum unit thereof. For example, a polypeptide or a peptide comprisinga minimum active unit (region or domain) related to the function ofProtein-X or Protein-Y is also provided in the present invention. Thepolypeptide or the peptide that has the aforementioned partial sequencecan be used, for instance, to inhibit the function of Protein-X and/orProtein-Y. More specifically, for example, the peptide being capable ofbinding to an NMDA receptor/2B subunit can inhibit the binding ofProtein-X to the NMDA receptor/2B subunit. In addition, for example, apeptide that is a partial peptide of Protein-X, or Protein-Y, andcontains a region that is involved in the interaction of both proteins,can inhibit the interaction. Alternatively, it can inhibit theamplification of the NMDA receptor mediated signal transduction that isgenerated in the presence of Protein-Y and Protein-X. Therefore, such apeptide can inhibit the promotion of the NMDA receptor mediated signaltransduction. In addition, the polypeptide or the peptide that has theaforementioned partial sequence is useful as a reagent in, for example,identifying a substance that controls the biological activity (bindingto the NMDA receptor/2B subunit, guanylate kinase activity, and/orinteraction with Protein-Y) of Protein-X or a polypeptide that has thesame biological activity as Protein-X. In addition, it is useful as areagent in, for example, identifying a substance that controls thebiological activity (such as interaction with Protein-X) of Protein-Y ora polypeptide that has the same biological activity as Protein-Y. If itis a peptide recognized immunologically, such as an epitope peptide, itcan be used to produce antibodies specific to Protein-X or Protein-Y, byusing it singly as an antigen, or by linking it to a carrier (forexample, keyhole limpet hemocyanin or egg-white albumin), as describedbelow.

Polynucleotides

In one aspect of the present invention, the polynucleotide of thepresent invention means a polynucleotide containing a nucleotidesequence coding for each of the polypeptides or peptides of the presentinvention, or a complementary nucleotide sequence thereof. For instance,it can be a polynucleotide comprising the nucleotide sequence coding forthe polypeptide having the amino acid sequence set forth in SEQ ID NO:1, 2, or 3 in the Sequence Listing, or a complementary nucleotidesequence thereof. Preferably, it is a polynucleotide comprising thenucleotide sequence set forth in SEQ ID NO: 4, 5, or 6 in the SequenceListing, or a complementary nucleotide sequence thereof. In the presentinvention, polynucleotides comprising complementary nucleotide sequencescan be referred to as complementary strands.

In another aspect, the present invention includes polynucleotides thathybridize under stringent conditions to the region corresponding to theaforementioned polynucleotide, preferably the polynucleotide comprisingthe nucleotide sequence set forth in SEQ ID NO: 4, 5, or 6 in theSequence Listing, or the complementary nucleotide sequence thereof.Hybridization conditions can follow the publications, for instance,Sambrook et al. eds. Molecular Cloning: A Laboratory Manual, SecondEdition, Cold-Spring Harbor Laboratory Press, 1989, and the like. Thesepolynucleotides do not necessarily need to be complementary sequences asfar as they hybridize to the target polynucleotide, preferably thepolynucleotide comprising the nucleotide sequence set forth in SEQ IDNO: 4, 5, or 6 in the Sequence Listing, or to the complementarynucleotide sequence thereof. For instance, it can be a polynucleotidehaving a nucleotide sequence homologous to the polynucleotide comprisingthe nucleotide sequence set forth in SEQ ID NO: 4, 5, or 6 in theSequence Listing, or the complementary sequence thereof, with at least70% or greater, preferably 80% or greater, more preferably 90% orgreater, and even more preferably 95% or greater. In this case, thenucleotide sequence of SAPAP-3 is not included in the present invention.In addition, the polynucleotides of the present invention includepolynucleotides or oligonucleotides having a nucleotide sequence ofconsecutive nucleotides of 10 or more, preferably 15 or more, and morepreferably 20 or more, which correspond to a specified region of thenucleotide sequence of the aforementioned polynucleotide, or thecomplementary nucleotide sequence thereof.

These polynucleotides provide genetic information useful inmanufacturing the polypeptides and the like of the present invention, orcan also be used as reagents or standards related to nucleic acids. Forinstance, they can be used as nucleic acids coding for Protein-X orProtein-Y, for example as probes or primers for detecting the gene orthe mRNA thereof, or as antisense oligonucleotides to control the geneexpression, and the like. In this sense, the polynucleotides and theoligonucleotides of the present invention include those corresponding totranslated-regions as well as those corresponding tountranslated-regions. Here, the screening for the polynucleotides codingfor the polypeptides of the present invention can be performed, forexample, by verifying the expressed protein using a well-known proteinexpression system and taking the biological activity thereof as anindicator. The biological activities to be used as indicators areexemplified by binding to an NMDA receptor/2B subunit; by the guanylatekinase activity;or by interaction with the polypeptide having the aminoacid sequence set forth in SEQ ID NO: 1 or 3 in the Sequence Listing.The interaction can be evaluated, for instance by measuring thepromotion of the NMDA receptor mediated signal transduction. Morespecifically, measurement can be performed by taking as an indicator thefunction of amplifying the NMDA receptor mediated signal transduction inthe presence of two types of polypeptides having the respective aminoacid sequences set forth in SEQ ID NO: 1 and 3 in the Sequence Listing.As examples of well-known protein expression systems, cell-free proteinexpression systems that use the technology of the ribosome systemderived from embryo or rabbit reticulocytes and the like (Nature, 1957,Vol. 179, p. 160-161) can be cited.

Recombinant Vectors

Recombinant vectors can be obtained by transfecting the polynucleotidesof the present invention into adequate vector DNAs. The vector DNAs tobe used can be selected appropriately depending on the type of host andpurpose of use. In addition to vector DNAs extracted from thosenaturally present, vector DNAs can be those that lack a part of the DNA,other than that required for multiplication. Examples include:chromosome, episome and virus derived vectors, such as bacterialplasmid-derived, bacteriophage-derived, transposon-derived, yeastepisome-derived, insertion element-derived, and yeast chromosomalelement-derived; virus-derived vectors such as those derived frombaculovirus, papovavirus, SV40, vaccinia virus, adenovirus, fowlpoxvirus, pseudorabies virus and retrovirus; as well as vectors combiningthe same, such as vectors derived from plasmid and bacteriophage geneticelements, including cosmid and phagemid and the like. In addition,expression vectors, cloning vectors, and the like can be used dependingon the purpose.

A recombinant vector has, as components, the target gene sequence andgene sequences that carry information regarding replication and control(such as a promoter, ribosome binding site, terminator, signal sequenceand enhancer), and is produced by combining them by using the well-knownmethods. As the method for transfecting the polynucleotides of thepresent invention into the previously mentioned vector DNAs, well-knownmethods can be adopted. For instance, a method can be used, whichcomprises: selecting an adequate restriction enzyme, treating DNA tocleave it at specific sites, mixing the DNA with vector DNA that hasbeen treated in the same way, and relegating with a ligase. Otherwise, adesired recombinant vector can also be obtained by ligating an adequatelinker to the target polynucleotide, and inserting it into themulticloning site of a vector that is suitable for the purpose.

Transformants

Transformants can be obtained by introducing the vector DNA containingaforementioned polynucleotide into a host being well-known to oneskilled in the art; the introducing methods are well-known to oneskilled in the art. The vector DNA introduced into a host can be onetype of vector DNA, or two or more types of vector DNA. For instance, itcan be the vector DNA containing the polynucleotide set forth in SEQ IDNO: 4 in the Sequence Listing; the vector DNA containing thepolynucleotide set forth in SEQ ID NO: 6; or both vector DNAs. Asexamples of host, Escherichia coli, yeast, Bacillus subtilis, insectcells, or animal cells and the like can be cited. As preferable systemswhen carrying out introduction of the gene, the method of integrationinto chromosomes can be cited if gene stability is to be considered;however, for simplicity, auto-replicating systems that use extranucleargenes can be employed. Introduction of a vector DNA into a host cell canbe carried out by a standard method, for instance, that described inSambrook et al. eds., Molecular Cloning: A Laboratory Manual, SecondEdition, Cold-Spring Harbor Laboratory Press, 1989. Concretely, calciumphosphate transfection, DEAE-dextran-mediated transfection,microinjection, cationic lipid-mediated transfection, electroporation,transduction, scrape loading, ballistic introduction, infection, and thelike can be cited.

The polypeptides or peptides of the present invention can be providedwhen an expression vector is used as the vector DNA for use intransformation of the host. The transformant (into which an expressionvector DNA containing aforementioned polynucleotide has been introduced)can be cultured under culture conditions and by culture methods that aresuitable for each host and well-known to one skilled in the art.Culturing can be performed by referring to the function (for examplebinding to an NMDA receptor/2B subunit, guanylate kinase activity, orinteraction with the polypeptide having the amino acid sequence setforth in SEQ ID NO: 1 or 3 in the Sequence Listing) of the polypeptideor peptide of the present invention that is expressed by thetransformant. Alternatively, culturing can be performed by referring tothe quantity of polypeptides or peptides generated inside or outside thehost; a passage culture or a batch culturing can also be performed byreferring to the quantity of transformant in the culture medium.

Collection and/or Purification of the Polypeptides and Peptides

The collection and/or purification of the polypeptides or peptides ofthe present invention from the culture media in which the transformantwas cultured, can be performed by referring to the function (forexample, binding to an NMDA receptor/2B subunit, guanylate kinaseactivity, or interaction with the polypeptide having the amino acidsequence set forth in SEQ ID NO: 1 or 3 in the Sequence Listing) of thepolypeptide or peptide. Fractionation methods based on differences insolubility using ammonium sulfate or alcohol and the like, gelfiltration, ion column chromatography, affinity chromatography, and thelike can be cited as methods of collection and/or purification, whichare used alone or in combination. Preferably, a method can be used, inwhich polypeptides or peptides are specifically adsorbed and collectedby using polyclonal antibodies or monoclonal antibodies that can beprepared against the polypeptides or the peptides based on theinformation of their amino acid sequences.

Antibodies

The antibodies can be produced by using the aforementioned polypeptidesor peptides as antigens. The antigen can be the polypeptides or thepeptides, or a fragment thereof; antigens can be composed of at least 8,preferably at least 10, more preferably at least 12, further preferably15 or more amino acids. In order to produce antibodies that are specificto the aforementioned polypeptides and/or peptides, it is preferable touse regions comprising amino acid sequences that are unique to thepolypeptides or peptides. This region of the amino acid sequence doesnot necessarily need to be homologous or identical to the amino acidsequences of the polypeptides or peptides. Sites that are exposed to theexterior of the tertiary structure of the protein are preferred. Even ifthe amino acid sequences at the exposed sites are discontinuous in theprimary structure, it suffices as far as they are amino acid sequencesthat are continuous on the exposed site. There are no particular limitson the antibodies, as long as they immunologically bind or recognize thepolypeptides and/or peptides. Whether or not this binding or recognitionoccurs is determined by the well-known antigen-antibody-bindingreaction.

In order to produce an antibody, a well-known method for antibodyproduction can be employed. For example, the antibody can be obtained byadministering the antigen to an animal with or without linking such to acarrier, in the presence or absence of an adjuvant, to induce immunitysuch as a humoral response and/or cell-mediated response. The carrier isnot limited in particular, as long as it does not exert a harmful effectby itself on the host and is capable of enhancing antigenicity.Cellulose, polymeric amino acids, albumin, keyhole limpet hemocyanin(KLH), and the like can be given as examples of carriers. Examples ofthe adjuvant can be Freund complete adjuvant (FCA), Freund incompleteadjuvant (FIA), Ribi (MPL), Ribi (TDM), Ribi (MPL+TDM), Bordetellapertussis vaccine, muramyl dipeptide (MDP), aluminum adjuvant (ALUM),and combinations thereof. Mouse, rat, rabbit, goat, horse, and the likeare suitable animals for immunization.

Polyclonal antibodies can be obtained from the serum of the animalssubjected to the aforementioned immunization means, by any suitablemethod for collecting antibodies. As a preferable means, the antibodiesare obtained by the immuno-affinity chromatography method.

Monoclonal antibodies can be produced by collecting antibody-producingcells (for instance, a lymphocyte derived from a spleen or a lymph node)from the animal that was subjected to the aforementioned immunization,followed by the introduction of a well-known transformation techniqueusing a permanently proliferating cell (for example, myeloma strain suchas P3X63Ag8 cells.) For example, an antibody-producing cell is fusedwith a permanently proliferating cell to produce a hybridoma by methodsbeing well-known to one skilled in the art, and then cloning of thehybridoma is performed, followed by selecting hybridoma that produces anantibody recognizing specifically the aforementioned polypeptides and/orpeptides. The antibody can be collected from the cultured solution ofthe hybridoma.

The polyclonal antibody or monoclonal antibodies thus obtained, whichcan recognize and bind to the aforementioned polypeptides and/orpeptides, can be used as a purification antibody, reagent, or labelingmarker for the polypeptides or peptides.

Among the polyclonal or monoclonal antibodies that recognize and bind tothe polypeptides and/or the peptides, for example, the antibodies thatdirectly bind to Protein-X and inhibit the biological activity thereof(for example, the binding activity to other proteins such as an NMDAreceptor, guanylate kinase activity, and/or interaction with Protein-Y)are useful in the elucidation, prevention, improvement, or treatment ofvarious diseases caused by an anomaly in Protein-X and/or the functionthereof. In addition, antibodies that directly bind to Protein-Y andinhibit the function thereof (for example at least the interaction withProtein-X) are useful in the elucidation, prevention, improvement, ortreatment of various diseases caused by an anomaly in Protein-Y and/orthe function thereof. For instance, antibodies that inhibit thepromotion of the NMDA receptor mediated signal transduction, wherein thepromotion was caused by the interaction of Protein-X with Protein-Y, canbe cited as examples of such antibodies. More specifically, antibodiesthat inhibit the amplification of the NMDA receptor mediated signaltransduction in the presence of both Protein-X and Protein-Y can becited.

Identification Method of Compounds

The polypeptides or peptides of the present invention, thepolynucleotides of the invention, the vectors into which thesepolynucleotides have been transfected, the transformants into whichthese vectors have been introduced, the protein synthesis system usingthese, or the antibodies that immunologically recognize thesepolypeptides and/or the peptides, provide a method that is effective inidentifying an inhibiting agent or a promoting agent of the functions ofthe aforementioned polypeptides or the aforementioned peptides, and/oridentifying an inhibiting agent or promoting agent of the expression ofthe aforementioned polynucleotides, when they are used alone or incombination. The identification method of the present invention can beestablished using pharmaceutical screening systems being well-known inthe art. According to the identification method of the invention,screening for an antagonist by drug design based on the tertiarystructure of the aforementioned polypeptide or peptide of the invention,screening for an inhibiting agent or a promoting agent of the expressionat the gene level using the protein synthesis system, or screening for asubstance recognized by an antibody using an antibody, and the like, ispossible.

For example, compounds can be identified which promote or inhibit theactivation of Protein-X and of polypeptides comprising substantially thesame amino acid sequence as Protein-X, by using polypeptides or peptidesderived from Protein-X; selecting conditions that allow for interactionsof a test compound with these polypeptides or peptides; bringing thesepolypeptides or peptides to contact with the compound under theseconditions; and detecting the presence, the absence, or the change in asignal generated by the interaction. Compounds can be identified whichpromote or inhibit the interaction of Protein-X with another proteinthat binds to the polypeptides or the peptides (for example, an NMDAreceptor), by using an assay system that measures the interaction;adding the test compound thereto; and detecting the presence, theabsence, or the change in a signal generated by the interaction. Theassay system can be established using screening systems being well-knownin the art. For instance, the method described below in Example 3 can beused. In addition, when using the guanylate kinase activity of Protein-Xas an indicator, compounds that promote or inhibit the guanylate kinaseactivity can be identified. Measurement of guanylate kinase activity canbe carried out according to the well-known methods (Cook, P. F., et al.,Biochemistry, 1982, Vol. 21 p. 5794 ff.; Wright, D. E., et al., Proc.Natl. Acad. Sci. USA, 1981, Vol. 78, p. 6048 ff.; Corbin, J. D., et al.,Methods in Enzymology, 1974, Vol. 38, p. 287).

In addition, using polypeptides or peptides derived from Protein-Y,compounds can be identified as described above, which inhibit or promotethe activity of Protein-Y and of a polypeptide having the samephysiological activity as that of Protein-Y. Compounds that inhibit orpromote the interaction of Protein-X with Protein-Y can be identifiedusing polypeptides or peptides derived from Protein-X and polypeptidesor peptides derived from Protein-Y, by using an assay system thatmeasures the interaction thereof; adding the test compound thereto; anddetecting the presence, the absence, or the change in a signal generatedby the interaction. Concretely, compounds can be identified whichinhibit or further promote the promotion of the NMDA receptor mediatedsignal transduction, wherein the promotion is caused by the interactionof Protein-X with Protein-Y, Alternatively, for example, compounds canbe identified which inhibit or promote the amplification of the NMDAreceptor mediated signal transduction in the presence of both Protein-Xand Protein-Y. The assay system can be established using screeningsystems beint well-known in the art. For example, the method describedbelow in Example 6 can be used.

In addition, compounds can be identified which interact with thepolynucleotides of the present invention and inhibit or promote theexpression thereof, by selecting conditions that allow the interactionof the polynucleotides with the test compound; bringing thepolynucleotides to contact with the test compound under the conditions;using a system that uses a signal and/or a marker that can detect theexpression of the polynucleotides; and detecting the presence, theabsence, or the change of the signal and/or marker. Compounds can beidentified which inhibit or promote the expression of thesepolynucleotides, such as the Protein-X or Protein-Y, by furtherincorporating the method of using transformants described below.

In addition, compounds can be identified which inhibit or promote theexpression of the polynucleotides (such as the Protein-X gene or theProtein-Y gene), by bringing the transformants of the present inventioninto contact with a test compound or a compound identified above underappropriate conditions; and detecting the presence, the absence, or thechange of the expression of the polynucleotides of the present invention(such as the Protein-X gene or the Protein-Y gene). The detection of thepresence, the absence, or the change of the expression of the Protein-Xgene or the Protein-Y gene can be performed simply by measuring thefunction of the expressed gene product. For example, the detection ofthe presence, the absence, or the variation of the expression of theProtein-X gene can be performed by measuring the guanylate kinaseactivity thereof, as an indicator. Alternatively, in order to detect thepresence, the absence, or the change of the expression of the Protein-Xgene or the Protein-Y gene, a system well-known in the art (which uses asignal or a marker for detection) can be introduced to detect thepresence, the absence, or the change of the signal or marker. Herein, asignal indicates an entity that is directly detected by the physicalproperties or the chemical properties thereof, and a marker indicates anentity that is detected indirectly taking the physical properties or thechemical properties thereof as indicators. Any substances well-known(such as Luciferase and green fluorescence protein (GFP) and the like)can be used for the signal: any substances well-known (such as areporter gene (such as the chloramphenicol acetyltransferase (CAT) gene)or a detection tag (such as His-tag)) can be used for the marker. Thesignals or markers can be ligated to the target gene sequence, andtransfected into a vector; the obtained vector can be used to produce atransformant by transforming a host cell therewith. In addition, methodsfor utilizing and for detecting these signals or markers are well-knownto those skilled in the art.

Compounds (that inhibit or promote the expression or function ofProtein-Y or Protein-Y) can be identified in an experimental systemwherein a fusion protein of Protein-X or Protein-Y with a His-tag isexpressed (for example in Escherichia coli) as shown in the examples, byadding the test compound to the experimental system and then detectingthe His-tag.

Another example is a method that comprises transforming a cell with theProtein-X gene, the Protein-Y gene, and the NMDA receptor gene forco-expression thereof, adding the test compound, and measuring theintracellular signal transduction due to the stimulation of the NMDAreceptor by a ligand using a well-known method in the art. Using such acell in which the aforementioned genes are co-expressed, compounds thatinhibit or promote the expression of Protein-X or Protein-Y can beidentified, in addition to compounds that inhibit or promote thefunction of Protein-X or Protein-Y.

The methods and experimental systems given as examples in the foregoingwere given for the sake of exemplifying by concrete description thereof.The compound identification methods and the experimental systems used inthe methods of the present invention are not restricted thereby.

A Controlling Agent of Interaction of Protein-Y with Protein-X, and aPharmaceutical Composition

In the present invention, it was found that Protein-X binds to an NMDAreceptor/2B subunit and amplifies the NMDA receptor mediated signaltransduction. Protein-Y also remarkably amplifies the membrane receptormediated signal transduction (for instance an NMDA receptor mediatedsignal transduction) in the presence of Protein-X. From the foregoing,it was inferred that, when a membrane receptor/ion channel (for example,an NMDA receptor), is stimulated by a ligand thereof or the like,Protein-Y accumulates in the vicinity of the receptor, and thenstabilizes the receptor and/or promotes the receptor mediated signaltransduction through an interaction with Protein-X. Protein-X andProtein-Y can be involved in physiological functions such asneurotransmitter release, wherein the function are triggered as a resultof the signal transduction mediated by a membrane receptor (for instanceof an NMDA receptor) that they are involved in. Therefore, by inhibitingor promoting the interaction of Protein-Y with Protein-X, physiologicalfunctions that Protein-Y and Protein-X are involved in (for example, atleast the NMDA receptor mediated signal transduction) can be controlled.In addition, NMDA receptor mediated signal transduction can becontrolled by inhibiting or promoting the binding of the NMDA receptorto Protein-X.

The present invention thus provides an agent and a method forcontrolling NMDA receptor mediated signal transduction, wherein theagent and the method utilizes a means for inhibiting or promoting thebinding of Protein-X to the NMDA receptor, and/or, a means forinhibiting or promoting the interaction of Protein-X with Protein-Y.Herein, a controlling agent is a collective term to designate aninhibiting agent, an antagonist, or a promoting agent and the like. Inaddition, a controlling method means an inhibiting method or a promotingmethod and the like. To promote the binding of Protein-X to an NMDAreceptor, for instance, Protein-X itself or the gene thereof can beused. To promote the interaction of Protein-X and/or of Protein-Y, theseproteins themselves or the genes thereof can be used. For instance,since the binding of Protein-X to an NMDA receptor or the interaction ofProtein-X with Protein-Y is promoted by overexpressing Protein-X, or byoverexpressing both Protein-X and Protein-Y, or by promoting thefunction thereof, the NMDA receptor mediated signal transduction canthereby be promoted. Inhibition of the binding of Protein-X to the NMDAreceptor, or inhibition of the interaction between Protein-X and/or ofProtein-Y is acceptable pratically, for instance by inhibiting theexpression of Protein-X and/or Protein-Y. An abnormal signal of amembrane receptor can be normalized by the inhibition of the expressionof the proteins, or the inhibition of the function thereof.

In addition, for instance, a compound identified by the aforementionedmethod can be cited as a means for inhibiting or promoting the bindingof Protein-X to the NMDA receptor, or the interaction of Protein-X withProtein-Y. The compound can be used as an inhibiting agent or apromoting agent of the binding of Protein-X to an NMDA receptor, or asan inhibiting agent or a promoting agent of the interaction of Protein-Xwith Protein-Y. Furthermore, it can be used as an inhibiting agent or apromoting agent of NMDA receptor mediated signal transduction. Morespecifically, the compound can be used as an inhibiting agent or apromoting agent of the amplification of NMDA receptor mediated signaltransduction in the presence of both Protein-X and Protein-Y of thepresent invention. A polypeptide or a peptide comprising the minimumactivity unit (region or domain) related to the function of thepolypeptide of the present invention, and being capable of inhibitingthe interaction of Protein-X with Protein-Y can be given as examples ofcompounds using as inhibitors. Such polypeptides or peptides can beidentified, for example, by designing based on the amino acid sequenceof Protein-Y, synthesizing by a peptide synthesis method beingwell-known in the art, and assaying by the aforementioned detectionmethod. In addition, a polypeptide comprising an amino acid sequencethat is substantially identical to the amino acid sequence set forth inSEQ ID NO: 1 in the Sequence Listing, and not interacting with thepolypeptide having the amino acid sequence set forth in SEQ ID NO: 3 inthe Sequence Listing, can be used as an inhibitor. In addition, anantibody that inhibits the interaction of Protein-Y with Protein-X canalso be given as one example of the aforementioned compound.

The aforementioned screened compounds and controlling agents can beused, singly or in combination thereof, as reagents or ingredients forpharmaceutical compositions. The compounds and controlling agents can beused in the elucidation of biological functions or diseases thatProtein-X and/or Protein-Y as well as the genes thereof are involved in.For example, the elucidation of the mechanism of the NMDA receptorsignal transduction, or the elucidation of neurodegenerative diseasesand diseases caused by an anomaly in the NMDA receptor mediated signaltransduction are contemplated.

In addition, the compounds identified by the aforementioned method canbe used as candidate compounds for a controlling agent (for instance, aninhibiting agent, an antagonist, or a promoting agent) of the activityof Protein-X or of a polypeptide that has the same biological activityas that of Protein-X, for example, of the binding activity to otherproteins such as an NMDA receptor, or of the guanylate kinase activity.In addition, they can also be used as candidate compounds for acontrolling agent (for instance, an inhibiting agent, an antagonist, ora promoting agent) related to the expression at the gene level ofProtein-X or of a polypeptide that has the same biological activity asthat of Protein-X. Alternatively, they can be used as candidatecompounds for a controlling agent, for instance, an inhibiting agent, anantagonist, or a promoting agent of the activity of Protein-Y or of apolypeptide that has the same biological activity as that of Protein-Y(for example, interaction with Protein-X). In addition, they can also beused as candidate compounds for a controlling agent (for instance, aninhibiting agent, an antagonist, or a promoting agent) related to theexpression at the gene level of Protein-Y or of a polypeptide that hasthe same biological activity as that of Protein-Y. For example,antisense oligonucleotides of the Protein-X gene or the Protein-Y genecan be given as examples of compounds that inhibit such an expression.The antisense oligonucleotide is obtained from the oligonucleotides(designed by referring to the nucleotide sequence of the Protein-X geneor the Protein-Y gene), by selecting oligomers that specifically inhibitthe expression of the Protein-X gene or the Protein-Y gene with anexpression system of the Protein-X gene or the Protein-Y gene. Herein,“specifically inhibit the expression of the Protein-X gene or theProtein-Y gene,” means to powerfully inhibit the expression of theProtein-X gene or the Protein-Y gene, while not inhibiting or weaklyinhibiting the expression of other genes.

The candidate compound screened in such a manner can be prepared as apharmaceutical composition by further selecting it while taking intoaccount the balance between biological usefulness and toxicity. Inaddition, the polypeptides or the peptides of the invention, thepolynucleotides of the invention or complementary strands thereof,vectors that contain the polynucleotides or complementary strandsthereof, as well as antibodies that immunologically recognize thepolypeptides or the peptides of the present invention, can be used inthe elucidation, diagnosis, prevention, improvement, or treatment ofvarious disease conditions caused by an anomaly in the expression orfunction of the polypeptide of the present invention, for example,neurodegenerative diseases. That is to say, the present inventionprovides a pharmaceutical composition that contains at least one amongthe aforementioned substances, for use singly or in combination thereof.

As an anomaly in the expression, excess or decrease in the expressioncan be cited. As anomalies in the functions, anomalies in the binding ofProtein-X to an NMDA receptor, or anomalies in the interaction ofProtein-X with Protein-Y, can be cited. These anomalies result inanomalies in NMDA receptor mediated signal transduction. Morespecifically, those result in excess, decrease, loss, or the like of theNMDA receptor mediated signal transduction. As neurodegenerativediseases, Alzheimer's disease, Parkinson's disease, and polyglutaminedisease, and the like can be cited: however neurodegenerative diseasesare not restricted to the above.

Protein-X possesses a single PDZ domain, while PSD-95 possesses three.This single PDZ domain was shown to also bind to an NMDA receptor/2Bsubunit. Therefore, Protein-X can probably support for PSD-95-likesignal transduction and exhibit binding selectivity to other receptorsand the like according to the number of PDZ domains. A scheme forenhancement of signals and normalization of abnormal signals that isderived from this inferred function can also be used.

One method for treating abnormal conditions related to the expression ofProtein-X and/or Protein-Y of the present invention, and/or a decrease,loss, or the like of the functions thereof is characterized byadministering Protein-X and/or Protein-Y, or a polypeptide having thesame functions as these proteins, or a therapeutically effective dose ofa compound that activates the genes coding therefore, together with apharmaceutically acceptable carrier, and thereby improving an abnormalcondition. Alternatively, using gene therapy, Protein-X and/or Protein-Yor a polypeptide having the same function as these proteins can beproduced within a cell of a subject. A well-known method can be used forthe gene therapy; for example, a replication-defective retroviral vectorcontaining the polynucleotide of the invention (that is produced bytransfecting the polynucleotide to the vector as described above) can beused in the gene therapy. In addition, for instance, using DNA or RNAthat codes for the target protein, cells derived from the subject can betreated ex vivo, for instance, using a retroviral plasmid vector, andthen the cells can be introduced into the subject.

When the expression of Protein-X and/or Protein-Y and/or the functionsthereof are excessive, an effective dose of the aforementionedinhibiting compound can be administered together with a pharmaceuticallyacceptable carrier into a subject to inhibit the function of theseproteins. For instance, the binding of Protein-X to an NMDA receptor,and/or the promotion of the NMDA receptor mediated signal transductionby the interaction of Protein-X with Protein-Y, thereby improving theabnormal condition. In addition, expression block can be used to inhibitthe expression of the gene coding for the endogenous polypeptide. Theexpression of the gene can be inhibited using an oligonucleotide thathas been generated in a cell or separately administered, which caninclude the antisense sequence of the gene. These oligonucleotides canbe designed and synthesized, based on the aforementioned polynucleotidesof the present invention. The oligonucleotides can be administered bythemselves; otherwise, a related oligomer can be expressed in vivo.

Functional defects such as excess, decrease, or loss of expressionand/or function of Protein-X and/or Protein-Y, lead to anomalies in thecontrol of physiological functions in which Protein-X and/or Protein-Yare involved, for instance, neurotransmitter release provoked as aresult of the membrane receptor mediated signal transduction, whichcauses disease conditions. Therefore, the present invention is useful inthe elucidation of the biological functions in which Protein-X and/orthe PJ1087 protein are involved, for instance, the elucidation of thecontrol of intracellular signal transduction and neurotransmitterrelease. In addition, the present invention is extremely useful for aprophylactic agent, an improving agent, or a therapeutic agent, as wellas a prevention method, an improvement method, or a treatment method fordiseases provoked by an anomaly in the control of intracellular signaltransduction or neurotransmitter release in which Protein-X and/orProtein-Y are involved. For instance, diseases caused by an anomaly ofthe NMDA receptor mediated signal transduction, or neurodegenerativediseases. Furthermore, it is extremely useful for the measurement methodused as a means for diagnosing the diseases.

Recently, observations were obtained in relation to the mechanism ofmemory recovery in the brain, in an experiment using a knockout mouse.That is, an NMDA receptor in the CA3 region of the hippocampus wasplaying an important role in memory recovery (Nakazawa, K., et al.,Science, 2002, Vol. 297, No. 5579, pp. 211-218). This function of theNMDA receptor, when normalized or facilitated, can prevent, improve, ortreat symptoms of decreased ability for memory in diseases related tomemory recovery such as Alzheimer's disease. From the forgoing, thepresent invention provides a medicament for use in the prevention, theimprovement, or the treatment of diseases related to memory recovery,such as Alzheimer's disease, to be developed.

It is preferable to prescribe the pharmaceutical compositions, thecontrolling agent, the prophylactic agents, the therapeutic agents, orthe improving agents of the present invention, in combination with anadequate medicinal carrier. Such a prescription can include atherapeutically effective dose of the polypeptides or the peptides ofthe invention, the polynucleotides of the invention or the complementarystrands thereof, the vectors comprising the polynucleotides or thecomplementary strands thereof, the antibodies that immunologicallyrecognize the polypeptides or the peptides of the invention, theaforementioned compounds, the aforementioned controlling agents,prophylactic agents, therapeutic agents, improving agents, orpharmaceutical compositions, and a pharmaceutically acceptable carrieror excipient. Examples of such a carrier can include physiologicalsaline, buffered physiological saline, dextrose, water, glycerol,ethanol, and mixtures thereof, but are not limited thereto. It isadequate to select a prescription that is suited to the administrationroute, and such prescriptions are well-known to those skilled in theart. These pharmaceutical compositions, controlling agents, prophylacticagents, therapeutic agents, or improving agents can be used alone ortogether with another compound or medicament that is needed in thetreatment.

The administration form of the pharmaceutical compositions, controllingagents, prophylactic agents, therapeutic agents, or improving agents ofthe present invention can be a systemic administration or a topicaladministration. One preferred mode of systemic administration isinjection, for example, venous injection. Other injection routes such assubcutaneous, intramuscular, or intraperitoneal injection can also beused. Other modes of administration can be oral administration, if anenteric formulation or capsule formulation can be suitably formulated.In addition, per mucosal administration or per cutaneous administrationusing a permeating agent such as bile salt, fusidic acid, or othersurfactants can also be used. Topical administration can be in the formsof plaster, paste, gel and similar form.

The required dosage range depends on the efficacy of the polypeptides orthe peptides of the invention, the polynucleotides of the invention orthe complementary strands thereof, the vectors comprising thepolynucleotides or the complementary strands thereof, the antibodiesthat immunologically recognize the polypeptides or the peptides of theinvention, the aforementioned compounds, controlling agents,prophylactic agents, improving agents, therapeutic agents, orpharmaceutical compositions, the administration route, thecharacteristics of the prescription, the characteristics of theconditions of the subject, and the assessment by the physician incharge. More specifically, an adequate dose is, for example, a rangebetween 0.1 μg and 100 μg per 1 kg of the body weight of the subject.However, these doses can be modified using general conventionalexperiments for optimization well-known in the field.

For formulations, formulation means that are well-known should beintroduced in accordance with the physical properties of each subjectsuch as a peptide, protein, oligonucleotide, antibody, or compound. Morespecifically, a formulation method for, for instance, powdered drug,pills, tablets, capsule formulations, aqueous solution formulations,ethanol solution formulations, liposome formulations, fat emulsions, orclathrates such as cyclodextrin can be used.

Powdered drugs, pills, capsule formulations, and tablets can bemanufactured using excipients such as lactose, glucose, sucrose, andmannitol; disintegrants such as starch and sodium alginate; lubricantssuch as magnesium, stearate, and talc; binders such as polyvinylalcohol, hydroxypropyl cellulose, and gelatin; surfactants such as fattyacid ester; and plasticizers such as glycerin. A solid pharmaceuticalcarrier can be used to manufacture a tablet or a capsule.

Suspending agents can be manufactured using water; sugars such assucrose, sorbitol, and fructose; glycols such as PEG; and oils.

Injectable solutions can be prepared using a carrier comprising a salinesolution, a glucose solution, or a mixture of salt water and glucosesolution.

Inclusion into liposomes can be performed, for instance, by adding asolution wherein the substance of interest has been dissolved in asolvent (such as ethanol), to a solution wherein phospholipids have beendissolved in an organic solvent (such as chloroform), then removing thesolvent by evaporation, adding a phosphate-buffered solution thereto,and after agitating, sonicating, and centrifugating, filtrating thesupernatant for recovery.

Creation of fat emulsions can be performed, for example, by mixing andheating the substance of interest, oil ingredients (vegetable oils suchas bean oil, sesame oil, and olive oil as well as MCT and the like),emulsifying agent (such as phospholipids) and the like to obtain asolution, then adding a required amount of water andemulsifying/homogenizing by an emulsifier (homogenizer, for instance,high-pressure-spray type or sonicating type and the like). In addition,this can also be lyophilized. Further, when preparing a fat emulsion, anemulsification helper can be added; examples of an emulsification helperinclude glycerin and sugars (for instance, glucose, sorbitol, fructose,and the like).

Creation of cyclodextrin clathrates can be performed, for instance, byadding a solution wherein cyclodextrin has been dissolved in water andthe like by heating to a solution wherein the substance of interest hasbeen dissolved in a solvent (such as ethanol), then cooling, filtratingthe deposition resulting from cooling, and dry-sterilizing. In so doing,the cyclodextrin to be used should be suitably selected fromcyclodextrins with different void diameters (α, β, and γ types)according to the size of the substance.

Measurement Method and Reagent for Diagnosis

The polypeptides or the peptides of the present invention, thepolynucleotides of the invention or the complementary strands thereof,or the antibodies that immunologically recognize the polypeptides or thepeptides can be used singly as diagnostic markers or reagents and thelike. When these are reagents, they can contain a substance such asbuffering solution, salt, stabilization agent, and/or antiseptic agent.The polypeptides or the peptides of the invention can be a cell in whichthese have been expressed by a genetic engineering method, cell-freesynthesis products, chemical synthesis products, or those prepared fromthe cells and biological samples, as well as those that are furtherpurified therefrom. In addition, if the functions of the polypeptides orpeptides of the present invention, for instance, binding to an NMDAreceptor, interaction with Protein-X, or interaction with Protein-Y, orfor example the amplification of the NMDA receptor mediated signaltransduction due to the interaction are not inhibited, the polypeptidesor the peptides of the invention may have another protein or peptide,for example such as β-galactosidase, immunoglobulin Fc fragment from IgGor the like, His-tag, Myc-tag, HA-tag or FLAG-tag, ligated to theN-terminal side or the C-terminal side, directly or indirectly via alinker peptide or the like, using genetic engineering techniques or thelike. In addition, if the functions of the polynucleotides of thepresent invention, for instance, expression of the encoded polypeptideand the functions of the expressed polypeptide are not inhibited, thepolynucleotides of the present invention may have a signal such asluciferase or green fluorescent protein (GFP), or a reporter gene suchas chloramphenicol acetyl transferase (CAT) gene ligated to the 5′ endside or the C-terminal side. In addition, the present invention alsoprovides a reagent kit comprising one or more containers that are filledwith one or more kinds of these reagents. Furthermore, for formulations,formulation means that are well-known in the art should be introduced,in accordance with each of a polypeptide, peptide, protein,polynucleotide, or antibody, and the like.

These reagents and kits can be used in the identification methods of thepresent invention. In addition, these reagents and kits can be used forquantitatively and/or qualitatively measuring the polypeptides orpeptides of the present invention, or polynucleotides coding for any oneof these. The measurement method can be established using methodswell-known to those skilled in the art. Examples of methods that can beused to measure the polypeptides or the peptides include radioimmunoassay, competitive binding assay, Western blot analysis, andELISA. In addition, the polynucleotides can be detected and quantifiedat the nucleic acid level, for example the RNA level, using, forinstance, amplification, PCR, RT-PCR, RNase protection, Northernblotting, and other hybridization methods.

The aforementioned reagents, reagent kits, and measurement methods canbe used in the detection method of diseases caused by an anomaly in theexpression of the polypeptides or peptides of the present invention, forinstance, Protein-X and/or Protein-Y, or the function thereof.Alternatively, they can be used in detection methods of variouspathologies caused by a mutation and the like of the DNA codingtherefor. Examples of the diseases include neurodegenerative diseases,such as Alzheimer's disease, Parkinson's disease, and polyglutaminedisease.

Examples of samples to be measured include cells, blood, urine, saliva,spinal fluid, tissue biopsy, or necropsy materials and the like derivedfrom an individual. In addition, the nucleic acids to be measured can beobtained from each of the aforementioned samples by nucleic acidpreparation methods well-known in the art. For the nucleic acid, genomicDNA can be used directly for detection, or it may be enzymaticallyamplified prior to analysis by PCR or other amplification methods. RNAor cDNA can be used in a similar manner. Deletions and insertions can bedetected in comparison with the normal genotype, based on the changes inthe sizes of the amplification products. Point mutations can beidentified by hybridizing the amplified DNA to a labeled DNA that codesfor the polypeptide of the invention.

That is to say, with a sample derived from an individual, the diseasecan be diagnosed, for instance, by detecting the presence of thecorresponding nucleic acid to the polynucleotide that codes for thepolypeptide or the peptide of interest using the interaction therewithand responsiveness thereto; determining the quantity present thereof;and/or identifying the variation thereof; as well as determining the invivo distribution of the polypeptide or peptide in the individual;and/or detecting the presence of the polypeptide or peptide; determiningthe quantity present thereof; and/or detecting the variation thereof.

Furthermore, the aforementioned diseases can be examined and diagnosedby qualitatively or quantitatively measuring the polypeptides orpeptides of the present invention or the nucleic acid coding for any oneof these, as diagnostics markers. That is to say, by using theaforementioned detection method, a method for examining or diagnosingthe diseases can further be performed.

EXAMPLES

The present invention will be further described in the followingexamples. However, the present invention is not limited to theseexamples.

Example 1

Isolation/identification of a Novel PSD Gene (Protein-X Gene)

Cloning was performed using a template cDNA that was prepared by reversetranscription polymerase chain reaction (RT-PCR) from humanbrain-derived mRNA based on clone hj02537, which was extracted as a genehaving a PDZ domain, an SH3 domain and a GK domain, via bioinformaticsfrom the cDNA analysis information database (containing information ofcDNA derived from human brain almost completely mRNA) of the Kazusa DNAResearch Institute, to verify the in vivo expression of the Protein-Xgene of the present invention.

First, human brain-derived mRNA was used to perform a reversetranscription reaction using the Super Script II kit (Gibco BRL). Aftermixing 0.2 μl of human brain-derived mRNA (1 μg/μl), 1 μl of oligo (dT)primer (0.5 μg/μl) and 10.8 μl of H₂O; the mixture was heated for 10minutes at 70° C., cooled on ice; and then 4 μl of 5× First strandbuffer, 2 μl of 0.1 M dithiothreitol (DTT) and 1 μl of 10 mMdeoxynucleotide trisphosphate (dNTP) mix were added. Next, 1 μl of SuperScript II (200 U/μl) was added, and the reaction was allowed to takeplace for 10 minutes at room temperature, for 50 minutes at 42° C., andfor 15 minutes at 70° C. to prepare cDNA.

Then, based on the nucleotide sequence of clone hj02537, the followingprimers were designed and synthesized. In the primer sequence, theunderlined ATG is a translation initiation codon. Primers Pr-HJf (SEQ IDNO: 7): 5′-GGGGACAAGTTTGTACAAAAAAGCAGGCTTAATGCCAGCTTTGTCAA CGG-3′ Pr-HJf(SEQ ID NO: 8): 5′-GGGGACCACTTTGTACAAGAAAGCTGGGTCCATCTTGGAGATAGAGC GG-3′

The Advantage 2 PCR kit (Clontech) was used for the polymerase chainreaction (PCR). After mixing 2 μl of 10× Advantage 2 PCR buffer, 0.2 μlof the above-mentioned Pr—HJf (53.09 pmol/μl), 0.3 μl of theabove-mentioned Pr—HJr (38.59 pmol/μl), 1 μl of cDNA prepared above, 1.6μl of 1.25 mM dNTP mix, 0.4 μl of 50× polymerase mix and 14.5 μl of H₂O(20 μl in total), PCR reaction was performed under the followingconditions.

Conditions for Carrying Out PCR

Pre-step (pre): 95° C. for 1 minute

Step 1: 95° C. for 30 seconds

Step 2: 68° C. for 2 minutes

(Steps 1-2 for 25 cycles)

Post-step (post): 68° C. for 5 minutes

The obtained PCR product was used to conduct cloning using the Gate waysystem (Invitrogen). After adjusting 16 μl of the PCR product to 100 μlwith TE buffer followed by adding 50 μl of 30% PEG8000/30 mM MgCl₂, themixture was centrifuged at room temperature (at 15,000 rpm for 10minutes). The pellet was washed with 70% ethanol, dried, and dissolvedin 30 μl of TE buffer. Thereafter, 1 μl of BP clonase reaction buffer, 1μl of the PCR product, 0.5 μl of entry vector (pDONR 201:150 ng/μl) and1.5 μl of TE buffer were mixed on ice (4 μl in total) followed by adding1 μl of BP clonase enzyme mix, and then the reaction was allowed to takeplace for 4.5 hours at 25° C. After the reaction, 0.5 μl of Proteinase Kwas added, and the reaction solution was incubated for 10 minutes at 37°C. to inactivate the enzyme mix. Competent cells JM109 were transformedwith 1 μl of this reaction solution, and pDONR 201/hj02537 #7 and pDONR201/hj02537 #9 were obtained. From the nucleotide sequences of theseclones, expression in the brain was verified.

Example 2

Expression of the Novel PSD (Protein-X)

In order to express Protein-X, an expression vector and an expressionsystem of Escherichia coli were established using the Gate way system(Invitrogen). The protein-coding region was amplified using theAdvantage 2 PCR kit using the clone hj02537 as a template. After mixing2 μl of 10× Advantage 2 PCR buffer, 0.2 μl of Pr—HJf (53.09 pmol/μl),and 0.3 μl of Pr—HJr (38.59 pmol/μl), which were constructed in Example1, 2 μl of clone hj02537 (1 ng/μl), 1.6 μl of 1.25 mM dNTP mix, 0.4 μlof 50× polymerase mix, and 13.5 μl of H2O (two samples of 20 μl in totalwere prepared), the PCR reaction was carried out in the same way as inExample 1.

After adjusting 35 μl of the PCR product to 100 μl with TE bufferfollowed by adding 50 μl of 30% PEG8000/30 mM MgCl₂, the mixture wascentrifuged (at 15,000 rpm for 10 minutes) at room temperature. Thepellet was washed with 70% ethanol, dried, and dissolved in 50 μl of TEbuffer. Thereafter, 1 μl of BP clonase reaction buffer, 1 μl of the PCRproduct, 0.5 μl of entry vector (pDONR 201:150 ng/μl), and 1.5 μl of TEbuffer were mixed on ice (4 μl in total). Then 1 μl of BP clonase enzymemix was added, and the reaction was allowed to take place overnight at25° C. After the reaction, 0.5 μl of Proteinase K was added, and thereaction solution was incubated for 10 minutes at 37° C., to inactivatethe enzyme mix. Competent cells JM109 were transformed with 1 μl of thisreaction solution, and pDONR 201/hj02537 #1 was obtained.

Next, using pDONR 201/hj02537 #1, a vector that expresses Protein-X as afusion protein with a 6× His-tag was constructed. After mixing 1 μl ofLR clonase reaction buffer, 1 μl of pDONR 201/hj02537 #1 (50 ng/μl), 0.5μl of 6× His-tag expression vector (pDEST17:150 ng/μl) and 1.5 μl of TEbuffer (4 μl in total) on ice, 1 μl of LR clonase enzyme mix was added,and then the reaction was allowed to take place for 2 hours at 25° C.After the reaction, 0.5 μl of Proteinase K was added, and the reactionsolution was incubated for 10 minutes at 37° C. to inactivate the enzymemix. Competent cells (BL21-SI) were transformed with 1 μl of thisreaction solution, and three clones (pDEST17/hj02537 #9, pDEST17/hj02537#10, and pDEST17/hj02537 #11) were obtained.

The three clones were tested for the presence or absence of induction byNaCl, for the production of the target protein and for preparation ofsoluble protein. Each Escherichia coli was cultured by agitatingovernight at 37° C. in 2 ml of LB medium containing ampicillin(hereinafter referred to as LB-Amp), to which NaCl (NaCl—) was notadded. After culturing a total volume of 3 ml of LB-Amp (NaCl—), towhich 300 μl of this pre-cultured solution had been added, underagitation for 2 hours at 37° C., 180 μl of 5 M NaCl was added thereto (afinal concentration of 0.3 M), and cultured under agitation for afurther 2 hours, at 37° C. As a control, 180 μl of H₂O was added insteadof NaCl, and the culture proceeded in the same way. Thereafter, twosamples of 1.2 ml of each cultured solution were prepared andcentrifuged (at 15,000 rpm for 10 minutes at 4° C.), and separated intosupernatants (hereinafter referred to as soluble fractions) and pellets.The pellets were suspended in 200 μl of 2% SDS/20 mM Tris (pH 7.4), or20 mM Tris (pH 7.4), and then sonicated and centrifuged again (for 10minutes at 15,000 rpm at 10° C. or 4° C.). This supernatant of thecentrifugation (hereinafter can be referred to as an insoluble fraction)was mixed with an equal volume of 2× sample buffer (2% SDS/50 mM Tris(pH 6.8)/30% glycerol/0.01% Bromophenol Blue (BPB)) containing 10%β-mercaptoethanol (ME); the mixture was boiled for 2 minutes, it wasthen subjected to SDS-polyacrylamide gel electrophoresis (SDS-PAGE) overa 7.5% polyacrylamide gel; and the proteins were detected by CoomassieBrilliant Blue (CBB) staining. In addition, the above-mentioned solublefraction was also subjected to SDS-PAGE to carry out protein detection.As a result, shown in FIG. 2, a band at approximately 65.8 kDa wasdetected, which is expected to be a fusion protein of the humanProtein-X with the 6× His-tag. It was considered that the fusion proteinwas present in small amounts in the soluble fraction of the culturedEscherichia coli mentioned above (FIG. 3), but was present in largeamounts in the insoluble fraction.

In order to test the expression of the fusion protein of Protein-X, withthe 6× His-tag in the soluble fraction, the culturing of theaforementioned Escherichia coli was performed at 25° C. Each Escherichiacoli was cultured overnight in 2 ml of LB-Amp (NaCl—), under agitationat 25° C. After culturing a total volume of 2.2 ml of LB-Amp (NaCl—), towhich 200 μl of this pre-cultured solution had been added, underagitation for 4 hours at 25° C., 132 μl of 5 M NaCl was added (a finalconcentration of 0.3 M), and culture under agitation at 25° C. for 2more hours. As a control, 132 μl of H₂O was added instead of NaCl, andcultured in the same way. Thereafter, two samples of 1 ml of thecultured solution were prepared and centrifuged (at 15,000 rpm for 10minutes at 4° C.), and separated into supernatants and pellets. Thepellets were suspended in 200 μl of 2% SDS/20 mM Tris (pH 7.4) and 20 mMTris (pH 7.4), and then sonicated and centrifuged again (at 15,000 rpmfor 10 minutes at 10° C. or 4° C.). The supernatants of thecentrifugation were mixed with an equal volume of 2× sample buffercontaining 10% β-ME; the mixture was boiled for 2 minutes and subjectedto SDS-PAGE over a 7.5% polyacrylamide gel; and the fusion proteins weredetected in the same way as described above. As shown in FIG. 4, thefusion protein of Protein-X with the 6× His-tag was also expressed inthe soluble fraction of the culture at 25° C.

The human Protein-X obtained as described above, based on the clonehj02537, was proven to comprise 576 amino acid residues as set forth inSEQ ID NO: 1 in the Sequence Listing, and possess a PDZ domain fromisoleucine (Ile) 139 to glycine (Gly) 219, an SH3 domain from methionine(Met) 231 to arginine (Arg) 296, and a GK domain from threonine (Thr)404 to asparagine (Asn) 500, in the amino acid sequence thereof.

Example 3

Binding of the Novel PSD (Protein-X) to an NMDA Receptor/2B Subunit

The binding activity of Protein-X to an NMDA receptor/2B subunit wasexamined using the Overlay method.

First, Escherichia coli (pDEST17/hj02537 #11) that expresses the fusionprotein of Protein-X with a 6× His-tag was cultured overnight in 2 ml ofLB-Amp (NaCl—) at 25° C. After culturing a total of 2.2 ml of LB-Amp(NaCl—), to which 200 μl of this pre-cultured solution had been added,under agitation for 4 hours at 25° C., 132 μl of 5 M NaCl was addedthereto (a final concentration of 0.3 M), and cultured under agitationfor 2 more hours at 25° C. to induce the production of Protein-X. Afterculturing, Escherichia coli was collected; 200 μl of extraction buffer(1% Triton X/10 mM Tris (pH 7.5)/150 mM NaCl/1 mM PMSF(phenylmethylsulfonylfluoride)) was added; and after sonication andincubation on ice for 20 minutes, centrifugation was performed (at15,000 rpm for 10 minutes at 4° C.). The supernatant of thecentrifugation was collected and used as a sample containing Protein-X.In addition, a fraction of the supernatant of the centrifugation wasused to carry out SDS-PAGE in the same way as described in Example 2, toverify the expression and the expressed amount of Protein-X by Westernblotting (FIG. 5), in order to determine the quantity of sample to use.The Western blot was carried out as described below. First, afterelectrophoresis, the proteins were transferred by the semi-dry method(for 1 hour at 100 mA) onto a wet PVDF transfer membrane, and themembrane was blocked with 5% skim milk/TBS-T for 1 hour at roomtemperature. Thereafter, a mouse anti-His antibody (Amersham PharmaciaBiotech) that was diluted 1000-fold with 5% skim milk/TBS-T was added,and allowed to react for 1 hour at room temperature. After the reaction,the transfer membrane was washed three times with TBS-T for 10 minutesat room temperature. Then a secondary antibody (anti-mouse IgGantibody-horseradish per oxidase (HRP) (Cell Signaling)) that wasdiluted 3000-fold with 5% skim milk/TBS-T was added, and was allowed toreact for 1 hour at room temperature. After that, a 10-minute wash withTBS-T was performed three times at room temperature. Thereafter, thesignal was detected using the ECL kit and Hyperfilm.

Escherichia coli pDEST17/hjPDZ (−) #2, a mutant that lacks the PDZdomain, which is the region that binds to an NMDA receptor/2B subunit,was prepared to be used as a negative control as described below.pDEST17/hjPDZ (−) #2 was cultured in 2 ml of LB-Amp (NaCl—) overnight at37° C. After culturing, a total of 2.2 ml of LB-Amp (NaCl—), to which200 μl of this pre-cultured solution had been added, under agitation for4 hours at 37° C., 132 μl of H₂O was then added thereto, and culturedunder agitation for a further 2 hours at 37° C., to induce theproduction of the Protein-X mutant. After culturing, Escherichia coliwas collected and treated as described above to prepare samplescontaining the Protein-X mutant. In addition, the expression and theexpressed amount thereof were determined in the same way as describedabove (FIG. 5).

Escherichia coli pDEST15/NMDA receptor #5, which expresses the fusionprotein of NMDA receptor/2B subunit with GST (hereinafter can bereferred to as GST-NMDA receptor/2B subunit), was prepared as describedbelow, and cultured overnight in 2 ml of LB-Amp (NaCl—) at 37° C. Afterculturing a total of 3 ml of LB-Amp (NaCl—), to which 300 μl of thispre-cultured solution had been added, under agitation for 4 hours at 37°C., 180 μl of 5 M NaCl was then added thereto (a final concentration of0.3 M), and cultured under agitation for 2 more hours at 37° C., toinduce the production of the fusion protein of NMDA receptor/2B subunitwith GST. As a control, H₂O was added instead of NaCl, and the cultureproceeded in the same way. After culturing, the Escherichia coli wascollected, 200 μl of 2% SDS/20 mM Tris (pH 7.5) was added, and theresulting solution was sonicated and centrifuged (at 15,000 rpm for 10minutes at 10° C.). The supernatant of the centrifugation was collectedand used as a sample containing an NMDA receptor/2B subunit. Inaddition, to verify the expression and the expressed amount of thetarget protein, a portion of the supernatant of the centrifugation wascollected and diluted one-fold and one-half-fold with 2% SDS/20 mM Tris(pH 7.5). Each diluted supernatant of the centrifugation and 3× samplebuffer (NEB) containing 10% β-ME were mixed at a proportion of 2:1, themixtures were boiled for 2 minutes and subjected to electrophoresis overa 5%-20% polyacrylamide gel. After electrophoresis, the proteins weretransferred onto a wet PVDF transfer membrane (Poly screen (NEN)) (for1.5 hours at 300 mA in a bath), and the membrane was blocked with 5%skim milk/TBS-T (TBS-T: 10 mM Tris (p H7.5)/150 mM NaCl/0.05% Tween 20)for 1 hour at room temperature. Thereafter, a goat anti-GST antibody(Amersham Pharmacia Biotech) that was diluted 1000-fold with 5% skimmilk/TBS-T, was added to the incubation solution, and allowed to reactfor 1 hour at room temperature. After the reaction, the transfermembrane was washed three times with TBS-T for 10 minutes at roomtemperature. Then a secondary antibody (anti-goat IgG antibody-HRP,Santa Cruz Biotechnology) that was diluted 2000-fold with 5% skimmilk/TBS-T was added, and allowed to react for 1 hour at roomtemperature. After that, a 10-minute wash with TBS-T was performed threetimes at room temperature. Thereafter, the signal was detected using theECL kit and Hyper Film Signal (Amersham Pharmacia Biotech) (FIG. 6).

In addition, in order to use as a negative control, Escherichia coliDH5α (derived from pGEX-4T-3 from Amersham Pharmacia Biotech) expressingonly GST was cultured in 8 ml of LB-Amp overnight at 37° C. Afterculturing a total of 22 ml of LB-Amp, to which 2 ml of this pre-culturedsolution had been added, for 1.5 hours at 37° C. until the value atOD600 reached to approximately 1.0, 42 μl of 500 mM IPTG (isopropyl1-thio-β-D-galactoside) was added thereto (a final concentration of 1mM), and cultured under agitation for 2 more hours at 37° C. Afterculturing, 1.5 ml samples were taken from each, and the bacteria werecollected. This Escherichia coli was treated as described above, and theobtained supernatant of the centrifugation was used as a sample. Inaddition, to verify the expression and the expressed amount of thetarget protein, a fraction of the supernatant of the centrifugation wascollected and diluted ×1/40, ×1/80, ×1/160, ×1/320, ×1/640 and ×1/1280with 2% SDS/20 mM Tris (pH 7.5). Using each diluted supernatant of thecentrifugation, the expression of GST was verified in the same way asdescribed above (FIG. 7).

Then, the binding activity of Protein-X to an NMDA receptor/2B subunitwas examined using the Overlay method. Concerning samples containing aGST-NMDA receptor/2B subunit (those induced with NaCl and thoseuninduced) or a GST prepared as described above, stock solutions wereused for samples containing a GST-NMDA receptor/2B subunit (induced anduninduced) and ×1/640 diluted solution with 2% SDS/20 mM Tris (pH 7.5)was used for sample containing GST, based on the results of verificationof the expressed amounts by Western blotting as described above (FIGS. 6and 7). Each sample and 3× sample buffer (NEB) containing 10% β-ME weremixed at a proportion of 2:1, and the mixtures were boiled for 2 minutesand then subjected to electrophoresis over a 5%-20% polyacrylamide gel.After electrophoresis, the proteins were transferred onto a wet PVDFtransfer membrane (for 1.5 hours at 300 mA in a bath), and the membranewas blocked with 5% skim milk/TBS-T for 2 hour at room temperature.

Subsequently, 3 ml of 1 mM PMSF/TBS-T was added to 20 μl of samplecontaining Protein-X, prepared as described above and 40 μl ofextraction buffer, or to 60 μl of sample containing the Protein-X mutantprepared as described above, to make a mixture. As a control, a mixtureobtained by adding 3 ml of 1 mM PMSF/TBS-T to 60 μl of extraction bufferwas used. Each mixture was used and allowed to react with each of thetransfer membranes for 2 hours at 4° C. to bind thereto. Thereafter, a5-minute wash with TBS-T was performed three times at room temperature,and the membranes were blocked again with 5% skim milk/TBS-T for 1 hourat room temperature. After blocking, a mouse anti-His antibody (AmershamPharmacia Biotech) was diluted 1000-fold with 5% skim milk/TBS-T andadded; and this composition was allowed to react for 1 hour at roomtemperature. After the reaction, the transfer membranes were washedusing TBS-T for 10 minutes at room temperature; the wash was performedthree times; then a secondary antibody (anti-mouse IgG antibody-HRP)(Cell Signaling) was diluted 3000-fold with 5% skim milk/TBS-T, thenadded and allowed to react for 1 hour at room temperature. After that, a10-minute wash with TBS-T was performed three times at room temperature.Thereafter, a signal was detected using the ECL kit and Hyper film.

As a result, a signal was detected at the position of the band for thefull-length GST-NMDA receptor/2B subunit (189.2 kDa) on the transfermembrane to which Protein-X was bound, which showed the binding ofProtein-X to the NMDA receptor/2B subunit. When the control and theProtein-X mutant were used, no signal was detected; that is to say, nobinding was observed (FIG. 8). No band was detected in any of thetransfer membranes for which binding to GST was tested, which showedthat no binding to GST was observed (FIG. 9). From the foregoing, it issuggested that Protein-X binds to an NMDA receptor/2B subunit via thePDZ domain thereof. In addition, FIG. 6 suggests that an NMDAreceptor/2B subunit is a protein that is susceptible to degradation.However, from the experimental result described above, wherein a signalwas detected only at the position of the band for the full-length NMDAreceptor/2B subunit (FIG. 8), as well as that GST is located at theN-terminus, the region that binds to Protein-X is expected to be at theC-terminus of the NMDA receptor/2B subunit, which agrees withinformation from the literature (Nagano, T. et al., Journal ofBiochemistry, 1998, Vol. 124, pp. 869-875). Proteins that possess a PDZdomain recognize sequences comprising three amino acid residues, where aserine (Ser) or a threonine (Thr) is followed by another amino acid thatis followed by a valine (Val) (SXV or TXV), which are present at theC-terminal of the target protein (tSXV motif). The C-terminal amino acidsequence of the NMDA receptor/2B subunit used in the present Example wasSDV.

Preparation of the Novel PSD (Protein-X) Mutant

The mutant, which lacks the PDZ domain that is the region for binding tothe NMDA receptor/2B subunit from the Protein-X, was prepared asdescribed in the following.

First, based on the nucleotide sequence of clone hj02537, the followingprimers were designed and synthesized. Primers Pr-Dpdz-F1 (SEQ ID NO:9): 5′-GGGGACAAGTTTGTACAAAAAAGCAGGCTGCAGCAAAGAGGAGACAC CATCAAA-3′

PCR was carried out using the Advantage 2 PCR kit with the clone hj02537as a template. After mixing 2 μl of 10× Advantage 2 PCR buffer, 0.2 μlof Pr-Dpdz-F1 (58.64 pmol/μl) and 0.3 μl of Pr—HJr (38.59 pmol/μl), 1 μlof clone hj02537 (1 ng/μl), 1.6 μl of 1.25 mM dNTP mix, 0.4 μl of 50×polymerase mix, and 14.5 μl of H₂O (20 μl in total), a PCR reaction wascarried out under the conditions described in Example 1.

Thereafter, 16 μl of the PCR product was adjusted to 50 μl with TEbuffer, and then 25 μl of 30% PEG8000/30 mM MgCl2 was added thereto. Themixture was centrifuged at room temperature (at 15,000 rpm for 10minutes), then, the pellet was washed with 70% ethanol, dried, anddissolved in 30 μl of TE buffer. Subsequently, 1 μl of BP clonasereaction buffer, 1 μl of the PCR product, 0.5 μl of entry vector (pDONR201: 150 ng/μl) and 1.5 μl of TE buffer were mixed on ice (4 μl intotal). 1 μl of BP clonase enzyme mix was added thereto, and then thereaction was allowed to take place for 2.7 hours at 25° C. After thereaction, 0.5 μl of Proteinase K was added, and the reaction solutionwas incubated for 10 minutes at 37° C., to inactivate the enzyme mix.Competent cells (DH5α) were transformed with 1 μl of this reactionsolution, and pDONR 210/hjPDZ(−) #2 was obtained.

Next, using pDONR 210/hjPDZ(−) #2, a vector that expresses a fusionprotein of a Protein-X mutant, that lacks the PDZ domain, with a 6×His-tag, was constructed. After mixing 1 μl of LR clonase reactionbuffer, 1 μl of pDONR 201/hjPDZ(−) #2 (50 ng/μl), 0.5 μl of 6× His-tagexpression vector (pDEST17:150 ng/μl) and 1.5 μl of TE buffer (4 μl intotal) on ice, 1 μl of LR clonase enzyme mix was added, and then thereaction was allowed to take place for 0.5 hours at 25° C. After thereaction, 0.5 μl of Proteinase K was added, and the reaction solutionwas incubated for 10 minutes at 37° C., to inactivate the enzyme mix.Competent cells (BL21-SI) were transformed with 1 μl of this reactionsolution, and three clones (pDEST17/hjPDZ(−) #1, pDEST17/hjPDZ(−) #2 andpDEST17/hjPDZ(−) #3) were obtained.

The three clones were tested for the presence or absence of induction byNaCl of the production of the target protein and also for thepreparation of a soluble protein. Each Escherichia coli was culturedovernight in 2 ml of LB-Amp (NaCl—) under agitation at 37° C. Afterculturing, a total volume of 3.3 ml of LB-Amp (NaCl—), to which 300 μlof this pre-cultured solution had been added, under agitation for 2hours at 37° C., 180 μl of 5 M NaCl was then added thereto (a finalconcentration of 0.3 M), and cultured under agitation for a further 2hours, at 37° C. As a control, 180 μl of H2O was added, and the cultureproceeded in the same way. Thereafter, two samples of 1.5 ml of eachcultured solution were prepared and centrifuged (at 15,000 rpm for 10minutes at 4° C.). The pellets were suspended in 150 μl of 2% SDS/20 mMTris (pH 7.4) and 20 mM Tris (pH 7.4), then sonicated and centrifugedagain (at 15,000 rpm for 10 minutes at 10° C. or 4° C.). Thesupernatants of the centrifugation were mixed with an equal volume of 2×sample buffer containing 10% β-mercaptoethanol. The mixture was boiledfor 2 minutes, subjected to SDS-PAGE on 5%-20% polyacrylamide gel, andthe target protein was detected in the same way as described in Example2. As a result shown in FIG. 10, a band at approximately 41.7 kDa wasdetected, which is expected to be a fusion protein of the humanProtein-X mutant protein with the 6× His-tag. The fusion protein wasobserved both in the soluble fraction and in the insoluble fraction ofthe cultured Escherichia coli mentioned above. In addition, theexpression thereof was also observed in the soluble fraction when theculture was performed at 25° C. (FIG. 11).

Preparation of an NMDA Receptor/2B Subunit

The preparation of an NMDA receptor/2B subunit was carried out usingclone fj07108 (Kazusa DNA Research Institute), which contains a portionthereof, as described below. Since clone fj07108 was lacking the 347amino acid residues on the N-terminal side, this region was firstsubcloned, by RT-PCT using human brain-derived mRNA as a template.

PCR was carried out using an Advantage 2 PCR kit in the same way as inExample 1, using a template cDNA that was reverse-transcribed from humanbrain-derived mRNA using superscript II with an oligo(dT) primer andrandom primers, and PCR primers (Pr-NMDAR2Bf and Pr-NMDAR2B (RV),described below) that were designed based on the nucleotide sequencedownstream from the AfIII site at position 352 of clone fj07108. The PCRprimers were designed taking into consideration of the region upstreamfrom the initiation codon and recombination. Thereafter, the PCRproduct, which ends were blunted and phosphorylated using the BKL kit(TaKaRa), was then introduced into pBluescript (SK—) (SmaI digested) togive pBS(SK—)/NMDAr—N #10 and pBS(SK—)/NMDAr—N #25. The identificationof the nucleotide sequence revealed that pBS(SK—)/NMDAr—N #10 had twomutations due to PCR, however, no amino acid substitution occurred.However, since the guanine (G) at 144 bp in the nucleotide sequence waslacking, a recombination was performed using pBS(SK—)/NMDAr—N #25 andexploiting the SmaI site present in the region downstream therefrom andthe BamHI site of pBluescript(SK—), and a new pBS(SK—)/NMDAr—N #3 wasobtained. The nucleotide sequence thereof was determined, which wasidentical to that of the NMDA receptor/2B subunit (Accession No:NM_(—)000843) registered in GenBank. Primers Pr-NMDAR2Bf (SEQ ID NO:10): 5′-TTTGGCTTCTACAAACCAAGGGAG-3′ Pr-NMDAR2B (RV) (SEQ ID NO: 11):5′-TCCAATCATACCATTCCAGGTTCCA-3′

Thereafter, the multicloning site (MCS) (pBluescript) of clone fj07108was digested with XhoI, which ends were then blunted using the Bluntingkit (TaKaRa), and was digested with AfIII. Subsequently, 1.5 kbpfragment, which was obtained by digesting the MCS of pBS(SK—)/NMDAr—N #3with XbaI and blunting the ends thereof followed by digesting withAfIII, was introduced into the clone fj07108 digested with AfIII asdescribed above to give pBS/NMDAr #2. pBS/NMDAr #2 is a vector thatcontains the full-length NMDA receptor/2B subunit.

The protein-coding region of the target gene was amplified using theAdvantage HF2 PCR kit (Clontech) with pBS/NMDAr #2 as a template. Theprimers were designed and synthesized, based on the full-lengthnucleotide sequence of the NMDA receptor/2B subunit. PCR was performedby mixing 2.5 μl of 10× Advantage HF2 PCR buffer, 0.4 μl of Pr-2Bf(52.94 pmol/μl), 0.6 μl of Pr-2Br (37.44 pmol/μl), 0.2 μl of pBS/NMDAr#2 (0.48 μg/μl), 2.5 μl of dNTP mix, 0.5 μl of 50× polymerase mix, and18.3 μl of H₂O (25 μl in total). Primers Pr-2Bf (SEQ ID NO: 12):5′-GGGGACAAGTTTGTACAAAAAAGCAGGCTTAATGAAGCCCAGAGCGG AG-3′ Pr-2Br (SEQ IDNO: 13): 5′-GGGGACCACTTTGTACAAGAAAGCTGGGTCCTGTTCCCTCACTCAGA C-3′

Conditions for Carrying Out PCR

Pre-step (pre): 95° C. for 1 minute

Step 1: 95° C. for 30 seconds

Step 2: 58° C. for 30 seconds

Step 3: 72° C. for 5 minutes

(Steps 1-3 for 2 cycles)

Step 4: 95° C. for 30 seconds

Step 5: 68° C. for 5 minutes

(Steps 4 and 5 for 20 cycles)

Post-step (post): 68° C. for 5 minutes

Then, the PCR product was subjected to electrophoresis (1% agarose gel).A band of the target gene of approximately 4.5 kbp was isolated. Afterthat, it was extracted with a Gen elute EtBr minus spin column (SIGMA),purified, dried, and then dissolved in 10 μl of TE buffer. Afteradjusting to 100 μl with TE buffer, 50 μl of 30% PEG8000/30 mM MgCl₂ wasadded. The mixture was centrifuged at room temperature (at 15,000 rpmfor 10 minutes), and then the pellet was washed with 70% ethanol, dried,and dissolved in 20 μl of TE buffer. Thereafter, 1 μl of BP clonasereaction buffer, 1 μl of the PCR product, 0.5 μl of entry vector (pDONR201:150 ng/μl), and 1.5 μl of TE buffer were mixed on ice (4 μl intotal); 1 μl of BP clonase enzyme mix was added thereto; and then thereaction was allowed to take place for 1.7 hours at 25° C. After thereaction, 0.5 μl of Proteinase K was added, and the reaction solutionwas incubated for 10 minutes at 37° C. to inactivate the enzyme mix.Competent cells (DH5α) were transformed with 1 μl of this reactionsolution, and pDONR 201/NMDA receptor #1 was obtained.

When the nucleotide sequence of pDONR 210/NMDA receptor #1 wasdetermined, mutations that were believed to be caused by PCR werepresent at several positions, which includes mutations accompanied byamino acid substitutions. Therefore, pDONR 210/NMDA receptor #1 wasdigested with MfeI and AatII to remove the mutation region, and afragment of approximately 3.4 kbp resulting from a digestion ofpBS/NMDAr #2 with MfeI and AatII was introduced thereto to obtain pDONR210/NMDA receptor #2. Since there were two positions in theprotein-coding region derived from clone fj07108, where the nucleotideswere different from those of the NMDA receptor/2B subunit registered inGenBank as Accession NO: NM_(—)000843 (the base at 2664 bp from theinitiation codon (C→T; Thr→Thr) and the base at 3499 bp (A→G; Ile→Val)),subcloning was performed by RT-PCR using human brain-derived mRNA andprimer sets of the primers Pr—R2B-5 and Pr—NMDAr—R8, and of Pr—R2B-7 andPr—NMDAr—R12. Then the nucleotide sequence of pBS/NMDAr #2 wasdetermined. As a result, the nucleotide sequence obtained from the humanbrain-derived mRNA, through the RT-PCR subcloning, was revealed to beidentical to that of clone fj07108. Primers Pr-R2B-5 (SEQ ID NO: 14):5′-TGAGAAGAATGAGGTCATGAGCA-3′ Pr-NMDAr-R8 (SEQ ID NO: 15):5′-GTCACAGTCGTAGAGCCCTA-3′ Pr-R2B-7 (SEQ ID NO: 16):5′-AGTTCCGAACAAAGGAGAACTCAC-3′ Pr-NMDAr-R12 (SEQ ID NO: 17):5′-GAGTTCTGACCCGTCACCGTCGTGG-3′

Next, a vector expressing a fusion protein of an NMDA receptor/2Bsubunit with GST was constructed. After mixing 1 μl of LR clonasereaction buffer, 1 μl of pDONR 201/NMDA receptor #2 (50 ng/μl), 0.5 μlof GST expression vector (pDEST15: 150 ng/μl), and 1.5 μl of TE buffer(4 μl in total) on ice, 1 μl of LR clonase enzyme mix was added thereto,and then the reaction was allowed to take place for 1 hour at 25° C.After the reaction, 0.5 μl of Proteinase K was added, and the reactionsolution was incubated for 10 minutes at 37° C., to inactivate theenzyme mix. Competent cells (BL21-SI) were transformed with 1 μl of thisreaction solution, and three clones (pDEST15/NMDA receptor #4,pDEST15/NMDA receptor #5, and pDEST15/NMDA receptor #6) were obtained.

The three clones were tested for induction by NaCl, for the productionof the target protein. Each Escherichia coli clone was culturedovernight in 2 ml of LB-Amp (NaCl—) under agitation at 37° C. Afterculturing a total of 3 ml of LB-Amp (NaCl—), to which 300 μl of thispre-cultured solution had been added, under agitation, for 4 hours at37° C., 180 μl of 5 M NaCl was added thereto (a final concentration of0.3 M), and cultured under agitation for a further 2 hours at 37° C. Asa control, 180 μl of H₂O was added, and the culture proceeded in thesame way. Thereafter, the cultured solution was centrifuged (at 15,000rpm for 10 minutes at 4° C.). The pellet was suspended in 200 μl of 2%SDS/20 mM Tris (pH 7.4), sonicated, and centrifuged (at 15,000 rpm for10 minutes at 10° C.). The supernatant of the centrifugation was mixedwith an equal volume of 2× sample buffer containing 10% β-ME. Themixture was boiled for 2 minutes and subjected to SDS-PAGE over a 5%-20%polyacrylamide gel. The target protein was detected using an anti-GSTantibody in the same way as described above. The results shown in FIG.12, a detected band at approximately 189.2 kDa, which is expected to bea fusion protein of the NMDA receptor/2B subunit with GST.

Example 4

Functional Analysis of the Novel PSD (Protein-X) in the Context of theNMDA Receptor

Protein-X was co-expressed with an NMDA receptor in a Xenopus oocyte,and the current response to the stimulation of the NMDA receptor by aligand was measured to examine the action of Protein-X on the signal ofthe NMDA receptor.

The following plasmids were used in the present test.

Plasmid DNA Containing the Novel PSD Gene (Protein-X Gene)

The cDNA corresponding to clone hj02537 extracted in Example 1 wasselected from a cDNA library prepared from the human immature myeloidcell line KG-1 according to a method described in the literature(Nomura, N., et al., DNA Research, 1994, Vol. 1, pp. 27-35), and used(FIG. 13). To date, no information regarding the sequence of this clonehas been disclosed.

Plasmid DNA Containing the NMDA Receptor/2B Subunit Gene

pBS/NMDAr prepared in Example 3 was used (FIG. 14).

Plasmid DNA Containing the NMDA Receptor I Gene

PCR was carried out using an Advantage 2 PCR kit in the same way as inExample 1, using a template cDNA that was reverse-transcribed from humanbrain-derived mRNA, using superscript II with an oligo(dT) primer andrandom primers, and PCR primers that were designed and synthesized basedon the nucleotide sequence of the NMDA receptor I. Thereafter, using theobtained PCR product, the pGEM-T Easy Vector systems (Promega) was usedto carry out TA cloning, and a pGEM-T Easy vector was obtained (FIG.15). The vector retains a 2658 bps translated region of the NMDAreceptor I between a position of 2 bp (CC) downstream from EcoRI, and aposition of 2 bp (GG) upstream from SpeI.

Plasmid DNA Containing the PSD-95 Gene

PCR was carried out using an Advantage 2 PCR kit in the same way as inExample 1, using a template cDNA that was prepared from humanbrain-derived mRNAs in the same way as described above, and PCR primersthat were designed and synthesized based on the nucleotide sequence ofthe PSD-95 gene. Thereafter, using the obtained PCR product, pGEM-T EasyVector systems (Promega) was used to carry out TA cloning, and thepGEM-T Easy vector was obtained (FIG. 16). The vector retains a 2657 bptranslated region of PSD-95 between a position of 3 bp (GAG) downstreamfrom SpeI, and a position of 4 bp (TTCC) upstream from EcoRI.

First, the plasmid DNA containing the NMDA receptor I gene was treatedwith SmaI, while the plasmid DNAs containing other genes were treatedwith NotI, leading to linearization of the plasmids, and thenphenol/chloroform purification was performed. Using the obtained DNA asa template, a MEGAscript kit (Ambion) was used according to the productinstructions to carry out RNA synthesis reaction. In so doing, SP6 wasused as an RNA polymerase for the PSD-95 gene, and T7 was used for othergenes. After completion of the RNA synthesis reaction, the template DNAwas removed by DNAase treatment. After the RNA synthesis reactionproduct was verified by 1% agarose gel electrophoresis,phenol/chloroform purification was performed. The synthesized RNA, afterpurification, was dissolved in sterile water, and prepared so as toobtain a total RNA concentration of 1 μg/μL. The RNA concentration wasdetermined by optical density.

The Xenopus was purchased from Shiki leda Chemicals Co., Ltd., fattedfor several weeks, and then oocytes were extracted. After the oocyteswere treated with collagenase and then cultured overnight, the NMDAreceptor RNA prepared as described above was micro-injected thereto by awell-known method.

In so doing, the NMDA receptor/2B subunit RNA and NMDA receptor I RNAwere mixed with a ratio of 1:2 and prepared so as to give 20 ng percell, and used. After injection, the oocytes were incubated for 48 hoursat 20° C. in a medium (115 mM NaCl, 2.5 mM KCl, 1.8 mM BaCl₂, and 10 mMHEPES, pH 7.2).

When co-expressing Protein-X or PSD-95 with the NMDA receptor, the NMDAreceptor RNA was injected in the same way as described above, and then10 ng per cell of Protein-X RNA or PSD-95 RNA was re-injected after 24hours. Thereafter, incubation proceeded in a culture medium for 24 hoursat 20° C.

Ligands were made to act on the NMDA receptor expressed in the oocytes;the influx of cations arising from the opening of the NMDA receptor ionchannel as a result thereof was measured as a variation of the currentby an electrophysiological measurement method. Herein, the measurementswere carried out by the two-electrode voltage clamp method according toa reference article (FEBS Letter, 1999, Vol. 458, pp. 295-298). The BAbuffer (115 mM NaCl, 2.5 mM KCl, 1.8 mM BaCl₂, and 10 mM HEPES, pH 7.2)was used as the measurement buffer, and the membrane potential was fixedat −70 mV.

A solution mixture of L-glutamic acid (Glu) and glycine (Gly) was usedfor ligands, which were made to act on the NMDA receptor by directinstillation to the oocytes. In addition, when quantitating themagnitude of the current, the value obtained by subtracting the meanvalue of the leak current prior to the stimulation from the maximumvalue of the inward current after stimulation by the ligands(hereinafter referred to as current variation), was used. Themeasurement of the current response was carried out using 5 to 6 cellsper experiment. Within the same experiment, large differences in thewaveform patterns due to the current responses were not observed betweeneach cell.

First, different concentrations of ligands (10 μM Glu+10 μM Gly, 100 μMGlu+10 μM Gly, and 1000 μM Glu+10 μM Gly) were made to act on theoocytes into which the NMDA receptor RNA was injected to verify ligandconcentration dependency. As a result it was demonstrated that theexperimental system was adequate for the measurement of the NMDAreceptor signal. In the present experiment, after the ligand solutionwas added, no removal by washing was performed, which made the wave formpattern different from the one in the reference bibliography (FEBSLetter, 1999, Vol. 458, pp. 295-298), and the inward current tended notto be transient, in particular when stimulation was performed at highconcentration of ligands. This is believed to be because the ligands andthe receptor stayed bound to each other, which kept the NMDA receptorchannel open, resulting in the continuation of the cationic influx.

Next, a solution mixture of 1000 μM Glu and 10 μM Gly was used asligands, to measure the current response in oocytes into which Protein-XRNA or PSD-95 RNA was co-injected with NMDA receptor RNA. The resultsare shown in FIG. 17 and Table 1. When Protein-X or PSD-95 wasco-expressed with the NMDA receptor, it was observed that the currentresponse to the ligand stimulation tended to be greater than that whenthe NMDA receptor was expressed by itself. In addition, when Protein-Xwas expressed, a current response approximately 1.5 times greater thanthat when PSD-95 was expressed was observed. TABLE 1 Current variation(μA) NMDA-R NMDA-R NMDA-R PSD-95 Protein-X #1 0.17 0.50 0.60 #2 0.070.27 0.81 #3 0.06 0.32 0.38 #4 0.13 0.31 0.57 #5 0.23 0.48 0.41 #6 — —0.54 Average 0.13 0.38 0.55 Standard 0.07 0.11 0.15 deviation

From the results of the above electrophysiological measurements, it wasrevealed that Protein-X forms an apparatus by binding to an NMDAreceptor/2B receptor, and that Protein-X is involved in the signalgenerated by the stimulation of the NMDA receptor, for instance, openingof the receptor ion channel and the accompanying cationic influx, and inthe promotion thereof.

Example 5

Isolation/Identification of the PJ01087 Gene (Protein-Y Gene)

The gene of the present invention was isolated and identified based onclone PJ01087, which was extracted by using the human brain-derivedlong-strand cDNA analysis data (bioinformatics) of the Kazusa DNAResearch Institute, as a gene having 96% homology to the ratPSD95/SAP90-associated protein 3 (SAPAP-3).

First, DHα competent cells were transformed with a pBluescript SK+vector containing clone PJ01087 for amplification, and the length of theinsert resulting from a NotI/Sal1 cut was verified to be approximately3.7 kb. PCR was performed with this as the template, using the primersdescribed below. The primers were designed and synthesized based on thenucleotide sequence of PJ01087, obtained from the information databasedescribed above. The PCR was carried out using an Advantage 2 PCR kit(Clontech) according to the user's manual. Primers Forward primer (SEQID NO: 18): 5′-GGGGACAAGTTTGTACAAAAAAGCAGGCTTAATGAGGGGTTACCATG GC-3′Reverse primer (SEQ ID NO: 19):5′-GGGGACCACTTTGTACAAGAAAGCTGGGCTAGTACGGGTGGAGAACC G-3′

As a result, a PCR product of approximately 2.9 kb containing thefull-length ORF of the PJ01087 clone was obtained. The PCR product wascloned into the entry vector pDONR™ 201, using the Gate way system(Invitrogen). The cloning was carried out according to the user's manualof the same system.

Expression of the PJ01087 Gene (the Protein-Y Gene)

Absence of error in the entire length of the nucleotide sequence of theinsert portion (approximately 2.9 kb) was verified in the obtainedvector. Thereafter, recombination into expression vector pDEST™ 17, thatmakes a protein possible to be expressed with a His-tag at theN-terminus in Escherichia coli, and into expression vector pDEST™ 15,that makes a protein possible to be expressed with GST-tag inEscherichia coli, was performed. The recombination was carried out usingthe Gate way system (Invitrogen), according to the user's manual.

Induction of the expression of protein coded by the Protein-Y gene wascarried out in Escherichia coli transformed with pDEST™ 17 or pDEST™ 15that contains Protein-Y gene. Pre-culture was performed under agitationat 37° C. in an LB medium containing ampicillin, until OD600 reached 0.5to 1.0. NaCl was then added to 300 μl of the pre-cultured solution toobtain a final concentration of 0.3 M (NaCl(+)), and culture underagitation proceeded for 4 hours at 37° C. to induce protein expression.As a control, H2O was added instead of NaCl, and culturing proceeded inthe same way (NaCl(−)). Thereafter, each cultured solution wascentrifuged to recover the bacteria. The bacteria were suspended inphosphate-buffered physiological saline (PBS), sonicated three times for10 seconds, centrifuged again (at 15,000 rpm for 10 minutes at 10° C. orat 4° C.), and separated into a supernatant (hereinafter referred to assoluble fraction) and a pellet. The pellet was re-suspended in PBS(referred to as insoluble fraction).

For each fraction, the protein was detected by Western blotting usingantibodies against His-Tag and GST-Tag. The results show that in bothtests using His-tag and GST-tag, an expression of approximately 115 kDaof the target protein and other proteins believed to be thedecomposition products thereof were observed in the insoluble fractionof the Escherichia coli induced with NaCl (FIGS. 18A and 18B). Onecell-free protein expression system, Rapid Translation System RTS5000(Roche Diagnostics), which is based on the protein expression system ofEscherichia coli, was used to examine protein expression; however thetarget protein was still found in the insoluble fraction.

The human protein thus obtained comprises 979 amino acid residues as setforth in SEQ ID NO: 3 in the Sequence Listing. In addition, the genecoding for the protein comprises 3705 bases as set forth in SEQ ID NO: 6in the Sequence Listing.

Example 6

Functional analysis of the PJ01087 gene product (the Protein-Y geneproduct) in the context of an NMDA receptor

The Protein-Y gene has 96% homology to SAPAP-3. Since SAPAP-3 is aprotein related to rat PSD-95/SAP90, it was anticipated that theProtein-Y gene product would form a complex with human PSD-95 and wouldbe involved in the constitution of the postsynaptic density. SincePSD-95 has been reported to be involved in the NMDA receptor mediatedsignal transduction, the Protein-Y gene and the PSD-95 gene wereco-expressed with the NMDA receptor gene in Xenopus oocytes, and thenthe current response to the stimulation of the NMDA receptor by a ligandwas measured to examine the effects of Protein-Y on the signal of theNMDA receptor. In so doing, a gene (SEQ ID NO: 4 in the SequenceListing) coding for the novel PSD protein (Protein-X) (SEQ ID NO: 1 inthe Sequence Listing) was used instead of PSD-95 and examined in thesame way as above. Protein-X possesses a PDZ domain, an SH3 domain, anda GK domain as PSD-95 does, and was inferred to bind to an NMDA receptorresulting in signal transduction. The method used in the test wasidentical to the method described in Example 4.

The following plasmids were used in the present test.

Plasmid DNA containing the PJ01087 gene (the Protein-Y gene)

The cDNA, corresponding to clone PJ01087 extracted in Example 5, wasselected from a cDNA library prepared from the human immature myeloidcell line KG-1 according to a method described in the literature(Nomura, N., et al., DNA Research, 1994, Vol. 1, pp. 27-35), and wasused in this experiment (FIG. 19).

Plasmid DNA containing the Protein-X gene

The plasmid DNA described in Example 4 was used (FIG. 13).

Plasmid DNA containing the NMDA receptor/2B subunit gene

The plasmid pBS/NMDAr (FIG. 14) prepared in Example 3 was used.

Plasmid DNA containing the NMDA receptor I gene

The plasmid DNA prepared in Example 4 was used (FIG. 15).

Plasmid DNA containing the PSD-95 gene

The plasmid DNA prepared in Example 4 was used (FIG. 16).

The effects of Protein-X and Protein-Y on the signal of the NMDAreceptor were tested by the same method as in Example 4.

When co-expressing Protein-Y, PSD-95, and/or Protein-X with the NMDAreceptor, the NMDA receptor RNA was injected into Xenopus oocytes in thesame way as described in Example 4, and then 10 ng RNA per cell ofProtein-Y, PSD-95, and/or Protein-X was re-injected after 24 hours.Thereafter, incubation proceeded in a culture medium for 24 hours at 20°C.

Ligands were made to act on the NMDA receptor expressed in the oocytes,and the influx of cations arising from the opening of the NMDA receptorion channel as a result thereof was measured by the same method asdescribed in Example 4. The results are shown in FIGS. 20 and 21. Asshown in Example 4, by co-expressing PSD-95 or Protein-X with the NMDAreceptor, the current response to the ligand stimulation was observed tobe greater than that when the NMDA receptor was expressed by itself. Inaddition, even by co-expressing Protein-Y and PSD-95 with an NMDAreceptor, the current response did not differ from that observed whenthe PSD-95 was expressed. Meanwhile, when Protein-Y was co-expressedwith Protein-X, a current response approximately seven to eight timesgreater than that when only Protein-X was expressed was obtained.

From the results of the above electrophysiological measurements, it wasrevealed that Protein-Y markedly amplifies the NMDA receptor mediatedsignal transduction in the presence of Protein-X. From the foregoing, itwas revealed that Protein-Y amplifies the intracellular signal generatedby the stimulation of the NMDA receptor, by an interaction withProtein-X that possesses a PDZ domain and is capable of binding to anNMDA receptor/2B subunit. Furthermore, it was revealed that it isinvolved in the opening of the NMDA receptor ion channel and thepromotion of the accompanying cation influx.

Example 7

Presence or absence of binding of the PJ01087 Protein (Protein-Y) toProtein-X

The following proteins were prepared to test the binding of Protein-Y toProtein-X by immunoprecipitation.

PJ01087 protein (Protein-Y)

200 μl of 1% Triton-X/20 mM Tris (pH 7.4) was added to a pelletcorresponding to 2 ml culture (see Example 5) of Escherichia coliwherein expression of the protein was carried out as a fusion proteinwith a His-tag. After sonication, centrifugation was performed for 5minutes at 15,000 rpm, and the supernatant thereof was used as thesample (His-PJ01087).

Protein-X

200 μl of 1% Triton-X/20 mM Tris (pH 7.4) was added to a pelletcorresponding to 2 ml culture of Escherichia coli, wherein expression ofa fusion protein of Protein-X with GST was carried out using clonehj02537 described in Example 1 by the same method as in Example 5, Aftersonication, centrifugation was performed at 15,000 rpm for 5 minutes andthe supernatant thereof was used as a sample (GST-hj02537). In addition,using Escherichia coli wherein a fusion protein of a mutant resultingfrom the deletion of the guanylate kinase domain from Protein-X with GSTwas expressed, the mutant protein was prepared in the same way(GST-hj(GK−)).

GST

As a control, GST alone was expressed, and a sample was prepared in thesame way (derived from pGEX-4T-3, Amersham Biosciences).

Treatment of each Protein Sample

To each of the aforementioned supernatants, 30 μl of Protein G-Sepharoseequilibrated with 1% Triton-X/20 mM Tris (pH 7.4) was added, and thenagitated for 1 hour at 4° C. After agitation, the samples werecentrifuged, and the supernatants thereof were used for binding assays.

Binding Assay

Each of the supernatants was dispensed as in (1)-(4), adjusted to 200 μlwith 1% Triton-X/20 mM Tris (pH 7.4) and agitated overnight at 4° C.

(1) 25 μl His-PJ01087+16 μl GST-hj02537

(2) 25 μl His-PJ01087+100 μl GST-hj(GK−)

(3) 25 μl His-PJ01087+4 μl GST

(4) 25 μl His-PJ01087

After adding 1 μl of anti-GST antibody (SIGMA) and agitating for 1 hourat 4° C., a further 30 μl of Protein G-sepharose was added thereto, andagitation proceeded for an additional 6 hours at 4° C. Thereafter, thesupernatant was removed by centrifugation, and Protein G-sepharose waswashed three times with 500 μl of 1% Triton-X/20 mM Tris (pH 7.4). Afterthe washes, 30 μl of 2× sample buffer (2% SDS/50 mM Tris (pH 6.8)/30%glycerol/0.01% bromophenol blue) containing 2-mercaptoethanol was addedto Protein G-sepharose and mixed. The samples were boiled for 2 minutes,subjected to electrophoresis on a 5%-20% polyacrylamide gel, andtransferred onto a PVDF membrane. Then Western blotting was performedwith anti-GST antibodies and anti-His-tag antibodies.

The result shows that Protein-X (GST-hj02537) and the Protein-X mutant(GST-hj (GK−)) were adsorbed onto Protein G-sepharose (Lanes 5 and 6 inFIG. 22). However, when Protein-Y (His-PJ01087) was reacted withProtein-X (GST-hj02537) or the Protein-X mutant (GST-hj (GK−)) toperform immunoprecipitation with an anti-GST antibody followed bydetecting with an anti-His-tag antibody, no band showing Protein-Y wasdetected. Thus, no result showing that binding of Protein-Y to Protein-Xexists could be obtained (lanes 5 or 6 in FIG. 23).

Possibilities for Industrial Use

Clone hj02537 was extracted as a gene having a PDZ domain, an SH3 domainand a GK domain, by way of bioinformatics from the human brain-derivedlong-strand cDNA library analysis information database of the Kazusa DNAResearch Institute, and the gene product (SEQ ID NO: 1) of this gene(SEQ ID NO: 4) was found to be a novel protein (Protein-X), which bindsto an NMDA receptor/2B subunit. Protein-X is believed to be, from thecharacteristics of the amino acid sequence thereof, of a guanylatekinase family, which is cell membrane associated proteins, as is PSD-95.In addition, Protein-X was found to be involved in the signal arisingfrom the stimulation of the NMDA receptor, and to have a promotingactivity thereof.

Similarly, clone PJ01087 was extracted as a gene having 96% homology tothe rat PSD95/SAP90-associated protein 3 (SAPAP-3). Then, the geneproduct (SEQ ID NO: 3) of this gene (SEQ ID NO: 6) was found to markedlyamplify the signal transduced from the NMDA receptor, by interactingwith Protein-X (SEQ ID NO: 1). The NMDA receptor mediated signaltransduction was amplified more strongly in the presence of bothProtein-X and Protein-Y than in the presence of Protein-X alone.

From the foregoing, the present invention is extremely useful forenabling the elucidation of biological functions or diseases in whichProtein-X and Protein-Y, as well as the genes thereof, are involved. Forexample, it is useful for elucidation of the mechanism of an NMDAreceptor mediated signal transduction, and elucidation of the mechanismof memory recall. Furthermore, it is useful for elucidation of diseasescaused by an anomaly in the NMDA receptor mediated signal transductionor an anomaly in memory recollection, for example neurodegenerativediseases such as Alzheimer's disease, Parkinson's disease, andpolyglutamine disease, as well as for prevention, treatment, orimprovement of these diseases.

Sequence Listing Free Text

SEQ ID NO: 7 in the Sequence Listing: designed polynucleotide based onthe sequence set forth in SEQ ID NO: 4 to be used as a primer

SEQ ID NO: 8 in the Sequence Listing: designed polynucleotide based onthe sequence set forth in SEQ ID NO: 4 to be used as a primer

SEQ ID NO: 9 in the Sequence Listing: designed polynucleotide based onthe sequence set forth in SEQ ID NO: 4 to be used as a primer

SEQ ID NO: 10 in the Sequence Listing: designed polynucleotide based onthe partial nucleotide sequence of the NMDA receptor/2B subunit to beused as a primer

SEQ ID NO: 11 in the Sequence Listing: designed polynucleotide based onthe partial nucleotide sequence of the NMDA receptor/2B subunit to beused as a primer

SEQ ID NO: 12 in the Sequence Listing: designed polynucleotide based onthe nucleotide sequence of the NMDA receptor/2B subunit to be used as aprimer

SEQ ID NO: 13 in the Sequence Listing: designed polynucleotide based onthe nucleotide sequence of the NMDA receptor/2B subunit to be used as aprimer

SEQ ID NO: 14 in the Sequence Listing: designed polynucleotide based onthe nucleotide sequence of the NMDA receptor/2B subunit to be used as aprimer

SEQ ID NO: 15 in the Sequence Listing: designed polynucleotide based onthe nucleotide sequence of the NMDA receptor/2B subunit to be used as aprimer

SEQ ID NO: 16 in the Sequence Listing: designed polynucleotide based onthe nucleotide sequence of the NMDA receptor/2B subunit to be used as aprimer

SEQ ID NO: 17 in the Sequence Listing: designed polynucleotide based onthe nucleotide sequence of the NMDA receptor/2B subunit to be used as aprimer

SEQ ID NO: 18 in the Sequence Listing: designed polynucleotide based onthe sequence set forth in SEQ ID NO: 6 to be used as a primer

SEQ ID NO: 19 in the Sequence Listing: designed polynucleotide based onthe sequence set forth in SEQ ID NO: 6 to be used as a primer

1. An agent for controlling N-methyl-D-aspartate receptor mediatedsignal transduction, wherein the agent inhibits or promotes the bindingof a polypeptide having the amino acid sequence set forth in SEQ ID NO:1, in the Sequence Listing, to the N-methyl-D-aspartate receptor, and/orinhibits or promotes the interaction of a polypeptide having the aminoacid sequence set forth in SEQ ID NO: 3, in the Sequence Listing, withthe polypeptide having the amino acid sequence set forth in SEQ ID NO:1, in the Sequence Listing.
 2. An agent for inhibitingN-methyl-D-aspartate receptor mediated signal transduction, wherein theagent inhibits binding of a polypeptide having the amino acid sequenceset forth in SEQ ID NO: 1, in the Sequence Listing, to theN-methyl-D-aspartate receptor, and/or inhibits interaction of apolypeptide having the amino acid sequence set forth in SEQ ID NO: 3, inthe Sequence Listing, with the polypeptide having the amino acidsequence set forth in SEQ ID NO: 1, in the Sequence Listing.
 3. An agentfor enhancing N-methyl-D-aspartate receptor mediated signaltransduction, wherein the agent promotes the binding of a polypeptidehaving the amino acid sequence set forth in SEQ ID NO: 1, in theSequence Listing, to the N-methyl-D-aspartate receptor, and/or promotesthe interaction of a polypeptide having the amino acid sequence setforth in SEQ ID NO: 3, in the Sequence Listing, with the polypeptidehaving the amino acid sequence set forth in SEQ ID NO: 1, in theSequence Listing.
 4. A method for controlling N-methyl-D-aspartatereceptor mediated signal transduction, wherein the method comprisesinhibiting or promoting the binding of a polypeptide having the aminoacid sequence set forth in SEQ ID NO: 1, in the Sequence Listing, to theN-methyl-D-aspartate receptor, and/or inhibiting or promoting theinteraction of the polypeptide having the amino acid sequence set forthin SEQ ID NO: 3, in the Sequence Listing with the polypeptide having theamino acid sequence set forth in SEQ ID NO: 1, in the Sequence Listing.5. A method for inhibiting N-methyl-D-aspartate receptor mediated signaltransduction, wherein the method comprises inhibiting the binding of apolypeptide having the amino acid sequence set forth in SEQ ID NO: 1, inthe Sequence Listing, to the N-methyl-D-aspartate receptor, and/orinhibiting the interaction of a polypeptide having the amino acidsequence set forth in SEQ ID NO: 3, in the Sequence Listing, with thepolypeptide having the amino acid sequence set forth in SEQ ID NO: 1, inthe Sequence Listing.
 6. A method for promoting N-methyl-D-aspartatereceptor mediated signal transduction, wherein the method comprisespromoting the binding of a polypeptide having the amino acid sequenceset forth in SEQ ID NO: 1, in the Sequence Listing, to theN-methyl-D-aspartate receptor, and/or by promoting the interaction of apolypeptide having the amino acid sequence set forth in SEQ ID NO: 3, inthe Sequence Listing, with the polypeptide having the amino acidsequence set forth in SEQ ID NO: 1, in the Sequence Listing.
 7. Apolypeptide selected from the following polypeptides: i. a polypeptidehaving the amino acid sequence set forth in SEQ ID NO: 1 or 2, in theSequence Listing; ii. a polypeptide containing the polypeptide set forthabove in (i); iii. a polypeptide having at least approximately 70%homology to the polypeptide set forth above in (i) at the amino acidsequence level and binding to an N-methyl-D-aspartate receptor/2Bsubunit; or iv) a polypeptide having a mutation, which is a deletion, asubstitution, an addition, or an insertion of one or more amino acids inthe amino acid sequence of the polypeptide set forth above in (i), andbinding to the N-methyl-D-aspartate receptor/2B subunit.
 8. Apolypeptide selected from the following polypeptides, wherein thepolypeptide binds to an N-methyl-D-aspartate receptor/2B subunit: i. apolypeptide having the amino acid sequence set forth in SEQ ID NO: 1 or2, in the Sequence Listing; ii. a polypeptide containing the polypeptideset forth above in (i); iii. a polypeptide having at least approximately70% homology to the polypeptide set forth above in (i) at the amino acidsequence level; or iv. a polypeptide having a mutation, which is adeletion, a substitution, an addition, or an insertion of one or moreamino acids in the amino acid sequence of the polypeptide set forthabove in (i).
 9. A polypeptide selected from the following polypeptides:i. a polypeptide having the amino acid sequence set forth in SEQ ID NO:3, in the Sequence Listing; ii. a polypeptide containing the polypeptideset forth above in (i); iii. a polypeptide having at least approximately70% homology to the polypeptide set forth above in (i) at the amino acidsequence level and interacting with a polypeptide having the amino acidsequence set forth in SEQ ID NO: 1, in the Sequence Listing; or iv. apolypeptide having a mutation, which is a deletion, a substitution, anaddition, or an insertion of one or more amino acids in the amino acidsequence of the polypeptide set forth above in (i) and interacting withthe polypeptide having the amino acid sequence set forth in SEQ ID NO:1, in the Sequence Listing.
 10. A polypeptide selected from thefollowing polypeptides, wherein the polypeptide interacts with apolypeptide having the amino acid sequence set forth in SEQ ID NO: 1, inthe Sequence Listing: i. a polypeptide having the amino acid sequenceset forth in SEQ ID NO: 3, in the Sequence Listing; ii. a polypeptidecontaining the polypeptide set forth above in (i); iii. a polypeptidehaving at least approximately 70% homology to the polypeptide set forthabove in (i) at the amino acid sequence level; or iv. a polypeptidehaving a mutation, which is a deletion, a substitution, an addition, oran insertion of one or more amino acids in the amino acid sequence ofthe polypeptide set forth above in (i).
 11. A polypeptide selected fromthe following polypeptides, wherein the polypeptide amplifiesN-methyl-D-aspartate receptor mediated signal transduction in thepresence of a polypeptide having the amino acid sequence set forth inSEQ ID NO: 1, in the Sequence Listing: i. a polypeptide having the aminoacid sequence set forth in SEQ ID NO: 3, in the Sequence Listing; ii. apolypeptide containing the polypeptide set forth above in (i); iii. apolypeptide having at least approximately 70% homology to thepolypeptide set forth above in (i) at the amino acid sequence level; oriv. a polypeptide having a mutation, which is a deletion, asubstitution, an addition, or an insertion of one or more amino acids inthe amino acid sequence of the polypeptide set forth above in (i).
 12. Apolypeptide, which binds to an N-methyl-D-aspartate receptor/2B subunitand does not interact with a polypeptide having the amino acid sequenceset forth in SEQ ID NO: 3, in the Sequence Listing, wherein thepolypeptide is: i. a polypeptide having at least approximately 70%homology to a polypeptide having the amino acid sequence set forth inSEQ ID NO: 1 or 2, in the Sequence Listing at the amino acid sequencelevel; or ii. a polypeptide having a mutation, which is a deletion, asubstitution, an addition, or an insertion of one or more amino acids inthe amino acid sequence of the polypeptide set forth above in (i).
 13. Apeptide comprising at least 5 consecutive amino acid residues within theamino acid sequence set forth in SEQ ID NO: 1 or 2, in the SequenceListing.
 14. A peptide comprising at least 5 consecutive amino acidresidues within the amino acid sequence set forth in SEQ ID NO: 1 or 2,in the Sequence Listing and binding to an N-methyl-D-aspartate receptor.15. A peptide comprising at least 5 consecutive amino acid residueswithin the amino acid sequence set forth in SEQ ID NO: 1, in theSequence Listing, and interacting with a polypeptide having the aminoacid sequence set forth in SEQ ID NO: 3, in the Sequence Listing.
 16. Apeptide comprising at least 5 consecutive amino acid residues within theamino acid sequence set forth in SEQ ID NO: 3, in the Sequence Listing.17. A peptide comprising at least 5 consecutive amino acid residueswithin the amino acid sequence set forth in SEQ ID NO: 3, in theSequence Listing and interacting with a polypeptide having the aminoacid sequence set forth in SEQ ID NO: 1, in the Sequence Listing. 18.The agent for controlling N-methyl-D-aspartate receptor mediated signaltransduction according to claim 1, wherein the agent comprises aneffective dose of at least one kind of polypeptide or peptide selectedfrom the polypeptides or peptides, of the following group: i. apolypeptide having the amino acid sequence set forth in SEQ ID NO: 1 or2, in the Sequence Listing; ii. a polypeptide containing the polypeptideset forth above in (i); iii. a polypeptide having at least approximately70% homology to the polypeptide set forth above in (i) at the amino acidsequence level and binding to an N-methyl-D-aspartate receptor/2Bsubunit; or iv. a polypeptide having a mutation, which is a deletion, asubstitution, an addition, or an insertion of one or more amino acids inthe amino acid sequence of the polypeptide set forth above in (i) andbinding to the N-methyl-D-aspartate receptor/2B subunit.
 19. The agentfor inhibiting N-methyl-D-aspartate receptor mediated signaltransduction according to claim 2, wherein the agent comprises aneffective dose of at least one kind of polypeptide or peptide selectedfrom the polypeptide which binds to an N-methyl-D-aspartate receptor/2Bsubunit and does not interact with a polypeptide having the amino acidsequence set forth in SEQ ID NO: 3, in the Sequence Listing, wherein thepolypeptide is: i. a polypeptide having at least approximately 70%homology to a polypeptide having the amino acid sequence set forth inSEQ ID NO: 1 or 2, in the Sequence Listing at the amino acid sequencelevel; or ii. a polypeptide having a mutation, which is a deletion, asubstitution, an addition, or an insertion of one or more amino acids inthe amino acid sequence of the polypeptide set forth above in (i). 20.The agent for promoting N-methyl-D-aspartate receptor mediated signaltransduction according to claim 3, wherein the agent comprises aneffective dose of at least one kind of polypeptide or peptide selectedfrom the polypeptides of the following group: i. a polypeptide havingthe amino acid sequence set forth in SEQ ID NO: 1 or 2, in the SequenceListing; ii. a polypeptide containing the polypeptide set forth above in(i); iii. a polypeptide having at least approximately 70% homology tothe polypeptide set forth above in (i) at the amino acid sequence leveland binding to an N-methyl-D-aspartate receptor/2B subunit; or iv. apolypeptide having a mutation, which is a deletion, a substitution, anaddition, or an insertion of one or more amino acids in the amino acidsequence of the polypeptide set forth above in i), and binding to theN-methyl-D-aspartate receptor/2B subunit.
 21. The method for controllingN-methyl-D-aspartate receptor mediated signal transduction according toclaim 4, wherein the method comprises utilizing at least one kind ofpolypeptide or peptide selected from the polypeptides or peptides fromthe following group: i. a polypeptide having the amino acid sequence setforth in SEQ ID NO: 1 or 2, in the Sequence Listing; ii. a polypeptidecontaining the polypeptide set forth above in (i); iii. a polypeptidehaving at least approximately 70% homology to the polypeptide set forthabove in (i) at the amino acid sequence level and binding to anN-methyl-D-aspartate receptor/2B subunit; or iv. a polypeptide having amutation, which is a deletion, a substitution an addition, or aninsertion of one or more amino acids in the amino acid sequence of thepolypeptide set forth above in (i), and binding to theN-methyl-D-aspartate receptor/2B subunit.
 22. The method for inhibitingN-methyl-D-aspartate receptor mediated signal transduction according toclaim 5, wherein the method comprises utilizing at least one kind ofpolypeptide or peptide selected from the polypeptide which binds to anN-methyl-D-aspartate receptor/2B subunit and does not interact with apolypeptide having the amino acid sequence set forth in SEQ ID NO: 3, inthe Sequence Listing, wherein the polypeptide is: i. a polypeptidehaving at least approximately 70% homology to a polypeptide having theamino acid sequence set forth in SEQ ID NO: 1 or 2, in the SequenceListing at the amino acid sequence level; or ii. a polypeptide having amutation, which is a deletion, a substitution, an addition, or aninsertion of one or more amino acids in the amino acid sequence of thepolypeptide set forth above in (i).
 23. The method for promotingN-methyl-D-aspartate receptor mediated signal transduction according toclaim 6, wherein the method comprises utilizing at least one kind ofpolypeptide or peptide selected from the polypeptides of the followinggroup: i. a polypeptide having the amino acid sequence set forth in SEQID NO: 1 or 2, in the Sequence Listing; ii. a polypeptide containing thepolypeptide set forth above in (i); iii. a polypeptide having at leastapproximately 70% homology to the polypeptide set forth above in (i) atthe amino acid sequence level and binding to an N-methyl-D-aspartatereceptor/2B subunit: or iv. a polypeptide having a mutation, which is adeletion, a substitution, an addition, or an insertion of one or moreamino acids in the amino acid sequence of the polypeptide set forthabove in (i) and binding to the N-methyl-D-aspartate receptor/2Bsubunit.
 24. A polynucleotide comprising a nucleotide sequence thatcodes for the polypeptide of claim 7, or a complementary nucleotidesequence thereof.
 25. A polynucleotide having the nucleotide sequenceset forth in SEQ ID NO: 4, or SEQ ID NO: 5, in the Sequence Listing, ora complementary nucleotide sequence thereof.
 26. A polynucleotide thathybridizes to the polynucleotide according to claim 24 under stringentconditions.
 27. A polynucleotide comprising a nucleotide sequence thatcodes for the polypeptide of claim 9, or a complementary nucleotidesequence thereof.
 28. A polynucleotide having a nucleotide sequence setforth in SEQ ID NO: 6, in the Sequence Listing, or a complementarynucleotide sequence thereof.
 29. A polynucleotide that hybridizes to thepolynucleotide according to claim 27 under stringent conditions.
 30. Arecombinant vector containing the polynucleotide according to claim 24.31. The recombinant vector according to claim 30, wherein therecombinant vector is a recombinant expression vector.
 32. A recombinantvector containing the polynucleotide according to claim
 27. 33. Therecombinant vector according to claim 32, wherein the recombinant vectoris a recombinant expression vector.
 34. A transformant that has beentransfected with the recombinant vector according to claim
 30. 35. Atransformant that has been transfected with the recombinant vectoraccording to claim
 32. 36. A transformant that has been transfected witha recombinant vector according to claim
 30. 37. A method formanufacturing the polypeptide according to claim 7, wherein the methodcomprises a step of culturing a transformant that has been transfectedwith a recombinant vector containing a polynucleotide comprising anucleotide sequence that codes for said polypeptide or a complementarynucleotide sequence thereof or a cell-free protein synthesis means thatuses the recombinant vector.
 38. A method for manufacturing thepolypeptide according to claim 9, wherein the method comprises a step ofculturing a transformant that has been transfected with a recombinantexpression vector containing a polynucleotide comprising a nucleotidesequence that codes for said polypeptide or a complementary nucleotidesequence thereof or a cell-free protein synthesis means that uses therecombinant vector.
 39. An antibody that immunologically recognizes thepolypeptide according to claim
 7. 40. An antibody that immunologicallyrecognizes the polypeptide according to claim 7, wherein the antibodyinhibits the function of said polypeptide.
 41. An antibody thatimmunologically recognizes the polypeptide according to claim
 9. 42. Anantibody that immunologically recognizes the polypeptide according toclaim 9, wherein the antibody inhibits the interaction of thepolypeptide having the amino acid sequence set forth in SEQ ID NO: 1, inthe Sequence Listing, with the polypeptide having the amino acidsequence set forth in SEQ ID NO: 3, in the Sequence Listing.
 43. Amethod for identifying a compound that interacts with the polypeptideaccording to claim 7 and inhibits or promotes the function thereof,wherein the method comprises utilizing the polypeptide, a polynucleotidecomprising a nucleotide sequence that codes for the polypeptide or acomplementary nucleotide sequence thereof, a recombinant vectorcontaining the polynucleotide, a transformant that has been transfectedwith the recombinant vector, or an antibody that immunologicallyrecognizes the polypeptide.
 44. A method for identifying a compound thatinteracts with the polypeptide according to claim 7 and inhibits orpromotes the function thereof, and/or a compound that interacts with apolynucleotide comprising a nucleotide sequence that codes for thepolypeptide or a complementary nucleotide sequence thereof and inhibitsor promotes the expression thereof, wherein the method comprisescontacting the compound with the polypeptide or the polynucleotide underconditions where the interaction of the compound with the polypeptide orthe interaction of the compound with the polynucleotide are allowed, anddetermining whether the compound interacts with the polypeptide or thepolynucleotide and inhibits or promotes the function of the polypeptideor the expression of the polynucleotide by detecting presence, absence,or variation of a signal which results from the interaction of thecompound with the polypeptide or the interaction of the compound withthe polynucleotide.
 45. A method for identifying a compound thatinteracts with the polypeptide according to claim 7 and inhibits orpromotes the function thereof, and/or a compound that interacts with apolynucleotide comprising a nucleotide sequence that codes for thepolypeptide or a complementary nucleotide sequence thereof and inhibitsor promotes the expression thereof, wherein the method comprisescontacting the compound with a transformant that has been transfectedwith a recombinant vector containing a polynucleotide comprising thenucleotide sequence that codes for the polypeptide, and determiningwhether the compound inhibits or promotes the expression or function ofthe polypeptide by utilizing a system that uses a signal and/or a markerbeing capable of detecting presence or absence of the expression or thefunction of the polypeptide to detect presence, absence, or variation ofthe signal and/or marker.
 46. A method for identifying a compound thatinhibits or promotes the binding of a polypeptide having the amino acidsequence set forth in SEQ ID NO: 1, in the Sequence Listing, to anN-methyl-D-aspartate receptor, wherein the method comprises utilizingthe polypeptide according to claim 7 a polynucleotide comprising anucleotide sequence that codes for the polypeptide or a complementarynucleotide sequence thereof a recombinant vector containing thepolynucleotide, a transformant that has been transfected with therecombinant vector, or an antibody that immunologically recognizes thepolypeptide.
 47. A method for identifying a compound that inhibits orpromotes the interaction of a polypeptide having the amino acid sequenceset forth in SEQ ID NO: 1, in the Sequence Listing, with a polypeptidehaving the amino acid sequence set forth in SEQ ID NO: 3, in theSequence Listing, wherein the method comprises utilizing the polypeptideaccording to claim 7, a polynucleotide comprising a nucleotide sequencethat codes for the polypeptide or a complementary nucleotide sequencethereof, a recombinant vector containing the polynucleotide, atransformant that has been transfected with the recombinant vector, oran antibody that immunologically recognizes the polypeptide.
 48. Acompound identified by the identification method according to claim 43.49. A compound that interacts with the polypeptide according to claim 7and inhibits or promotes the function thereof.
 50. A compound thatinteracts with the polynucleotide according to claim 24 and inhibits orpromotes the expression thereof.
 51. A compound that interacts with thepolypeptide according to claim 9 and inhibits or promotes theinteraction of the polypeptide with a polypeptide having the amino acidsequence set forth in SEQ ID NO: 1, in the Sequence Listing.
 52. Acompound that interacts with the polypeptide according to claim 9 andinhibits or promotes the amplification of the N-methyl-D-aspartatereceptor mediated signal transduction in the presence of the polypeptideand a polypeptide having the amino acid sequence set forth in SEQ ID NO:1, in the Sequence Listing.
 53. A compound that interacts with thepolynucleotide according to claim 27 and inhibits or promotes theexpression thereof.
 54. A pharmaceutical composition comprising aneffective dose of the polypeptide according to claim 7, a peptidecomprising at least 5 consecutive amino acid residues within the aminoacid sequence set forth in SEQ ID NO: 1, 2, or 3, in the SequenceListing, a polynucleotide comprising a nucleotide sequence that codesfor the polypeptide or a complementary nucleotide sequence thereof, arecombinant vector containing the polynucleotide, a transformant thathas been transfected with the recombinant vector, or an antibody thatimmunologically recognizes the polypeptide.
 55. An agent for preventing,treating, or improving a disease caused by an anomaly in theN-methyl-D-aspartate receptor mediated signal transduction, wherein theagent comprises an effective dose of the polypeptide according to claim7, a peptide, comprising at least 5 consecutive amino acid residueswithin the amino acid sequence set forth in SEQ ID NO: 1, 2, or 3, inthe Sequence Listing, a polynucleotide comprising a nucleotide sequencethat codes for the polypeptide or a complementary nucleotide sequencethereof, a recombinant vector containing the polynucleotide, atransformant that has been transfected with the recombinant vector, oran antibody that immunologically recognizes the polypeptide.
 56. Anagent for preventing, treating, or improving a disease caused by ananomaly in memory recall, wherein the agent comprises an effective doseof the polypeptide according to claim 7, a peptide comprising at least 5consecutive amino acid residues within the amino acid sequence set forthin SEQ ID NO: 1, 2, or 3, in the Sequence Listing, a polynucleotidecomprising a nucleotide sequence that codes for the polypeptide or acomplementary nucleotide sequence thereof, a recombinant vectorcontaining the polynucleotide, a transformant that has been transfectedwith the recombinant vector, or an antibody that immunologicallyrecognizes the polypeptide.
 57. An agent for preventing, treating, orimproving a neurodegenerative disease, wherein the agent comprises aneffective dose of the polypeptide according to claim 7, a peptidecomprising at least 5 consecutive amino acid residues within the aminoacid sequence set forth in SEQ ID NO: 1, 2, or 3, in the SequenceListing, a polynucleotide comprising a nucleotide sequence that codesfor the polypeptide or a complementary nucleotide sequence thereof, arecombinant vector containing the polynucleotide, a transformant thathas been transfected with the recombinant vector, or an antibody thatimmunologically recognizes the polypeptide.
 58. An agent for preventing,treating, or improving Alzheimer's disease, wherein the agent comprisesan effective dose of the polypeptide according to claim 7, a peptidecomprising at least 5 consecutive amino acid residues within the aminoacid sequence set forth in SEQ ID NO: 1, 2, or 3, in the SequenceListing, a polynucleotide comprising a nucleotide sequence that codesfor the polypeptide or a complementary nucleotide sequence thereof, arecombinant vector containing the polynucleotide, a transformant thathas been transfected with the recombinant vector or an antibody thatimmunologically recognizes the polypeptide.
 59. A method for preventing,treating, or improving a disease caused by an anomaly in theN-methyl-D-aspartate receptor mediated signal transduction, wherein themethod comprises administering at least one polypeptide according toclaim 7, the peptide comprising at least 5 consecutive amino acidresidues within the amino acid sequence set forth in SEQ ID NO: 1, 2, or3, in the Sequence Listing, a polynucleotide comprising a nucleotidesequence that codes for the polypeptide or a complementary nucleotidesequence thereof, a recombinant vector containing the polynucleotide, atransformant that has been transfected with the recombinant vector, oran antibody that immunologically recognizes the polypeptide.
 60. Amethod for preventing, treating, or improving a disease caused by ananomaly in memory recall, wherein the method comprises administering atleast one polypeptide according to claim 7, a peptide comprising atleast 5 consecutive amino acid residues within the amino acid sequenceset forth in SEQ ID NO: 1, 2, or 3, in the Sequence Listing, apolynucleotide comprising a nucleotide sequence that codes for thepolypeptide or a complementary nucleotide sequence thereof, arecombinant vector containing the polynucleotide, a transformant thathas been transfected with the recombinant vector, or an antibody thatimmunologically recognizes the polypeptide.
 61. A method for preventing,treating, or improving a neurodegenerative disease, wherein the methodcomprises administering at least one polypeptide according to claim 7, apeptide comprising at least 5 consecutive amino acid residues within theamino acid sequence set forth in SEQ ID NO: 1, 2, or 3, in the SequenceListing, a polynucleotide comprising a nucleotide sequence that codesfor the polypeptide or a complementary nucleotide sequence thereof, arecombinant vector containing the polynucleotide, a transformant thathas been transfected with the recombinant vector, or an antibody thatimmunologically recognizes the polypeptide.
 62. A method for preventing,treating, or improving Alzheimer's disease, wherein the method comprisesadministering at least one polypeptide according to claim 7, a peptidecomprising at least 5 consecutive amino acid residues within the aminoacid sequence set forth in SEQ ID NO: 1, 2, or 3, in the SequenceListing, a polynucleotide comprising a nucleotide sequence that codesfor the polypeptide or a complementary nucleotide sequence thereof, arecombinant vector containing the polynucleotide, a transformant thathas been transfected with the recombinant vector, or an antibody thatimmunologically recognizes the polypeptide.
 63. A method forquantitatively or qualitatively measuring the polypeptide according toclaim 7, or a polynucleotide comprising a nucleotide sequence that codesfor the polypeptide.
 64. A reagent kit comprising at least onepolypeptide according to claim 7, a peptide comprising at least 5consecutive amino acid residues within the amino acid sequence set forthin SEQ ID NO: 1, 2, or 3, in the Sequence Listing, a polynucleotidecomprising a nucleotide sequence that codes for the polypeptide or acomplementary nucleotide sequence thereof, a recombinant vectorcontaining the polynucleotide, a transformant that has been transfectedwith the recombinant vector, or an antibody that immunologicallyrecognizes the polypeptide.
 65. A method for controlling humanN-methyl-D-aspartate receptor mediated signal transduction, wherein themethod comprises inhibiting or promoting the interaction of thepolypeptide having the amino acid sequence set forth in SEQ ID NO: 3 inthe Sequence Listing with the polypeptide having the amino acid sequenceset forth in SEQ ID NO:
 1. 66. A method for controlling humanN-methyl-D-aspartate receptor mediated signal transduction, wherein themethod comprises inhibiting or promoting the binding of a polypeptidehaving the amino acid sequence set forth in SEQ ID NO: 1 in the SequenceListing to a human N-methyl-D-aspartate receptor.
 67. A polypeptidehaving the amino acid sequence set forth in SEQ ID NO: 2 in the SequenceListing.
 68. A polypeptide having the amino acid sequence set forth inSEQ ID NO: 2 in the Sequence Listing, wherein the polypeptide binds to ahuman N-methyl-D-aspartate receptor/2B subunit.
 69. A polypeptide havingthe amino acid sequence set forth in SEQ ID NO: 3 in the SequenceListing or a polypeptide containing the polypeptide.
 70. A polypeptidehaving the amino acid sequence set forth in SEQ ID NO: 3 in the SequenceListing or a polypeptide containing the polypeptide, wherein thepolypeptide interacts with a polypeptide having the amino acid sequenceset forth in SEQ ID NO: 1.